Nucleic acid vaccines against herpes simplex virus type 2: compositions and methods for eliciting an immune response

ABSTRACT

Herpes Simplex Virus-2 (HSV-2) infection is a major health concern. Highly effective vaccines and immunogenic compositions against HSV-2 which can be used therapeutically or prophylactically, are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US2012/066241, filed Nov. 21, 2012, which claims the benefit of U.S.Provisional Application No. 61/563,507, filed Nov. 23, 2011, thecontents of each of which are hereby incorporated herein in theirentirety.

SEQUENCE LISTING

In accordance with 37 CFR 1.52(e)(5), a Sequence Listing in the form ofan ASCII text file (entitled “Sequence_Listing.txt,” created on Nov. 21,2012, and 323 KB in size) is incorporated herein by reference in itsentirety.

BACKGROUND

Herpes simplex virus type 2 (HSV-2) is the leading cause of genitalherpes. HSV-2 is most often transmitted by sexual contact, and infectionwith the virus typically leads to recurring outbreaks of lesions on thegenitals and perianal regions, combined with shedding of virus into thegenital tract. Viral shedding can also occur in the absence of lesionsor other symptoms. HSV-2 also establishes latency in sensory ganglia.HSV-2 infection causes physical discomfort and psychosexual morbidity inaffected patients, and introduces additional health risks. Inparticular, patients infected with HSV-2 are at increased risk forcontracting HIV, and pregnant mothers infected with HSV-2 can verticallytransmit HSV-2 to their fetuses. In immunocompromised individuals or inneonates, HSV-2 infections can be fatal. Currently, there is no cure forHSV-2 infection.

HSV-2 infection is widespread, with one study estimating that nearly 20%of the population worldwide is infected (Looker et al., 2008, Bulletinof the World Health Organization, October 2008, 86(10)). More women thanmen are infected, and the prevalence of the disease increases with age.High numbers of adolescents diagnosed with HSV-2 indicate that theprevalence across the population will continue to rise, as HSV-2infection is lifelong.

Treatment options for HSV-2 symptoms are limited. Antiviral therapy,using compounds such as famciclovir, valaciclovir, or aciclovir, limitsthe duration of symptoms and, in some cases, speeds healing of lesionsand reduces incidence of viral shedding. Antiviral drugs are notcurative, however, and do not prevent recurrence of outbreaks or clearthe virus completely. In addition, use of antiviral drugs requirespatients to recognize symptoms of HSV-2 infection, then obtain aconfirmative diagnosis, and ultimately, comply with the antiviralregimen. These requirements may be untenable in regions of the worldwhere antiviral drugs are not readily available. In addition, patientsare often unaware that they are infected, either because they do notpresent symptoms, or because the symptoms of the initial infectionsubside, suggesting recovery from the disease.

To address the medical and social problems associated with HSV-2, it ishighly desirable to develop pharmaceutical compositions to inhibit orcounteract infection by HSV-2. An effective composition may be used toelicit an enhanced immune response against HSV-2, thereby preventinginitial infection, blocking the ability of the virus to establishlatency in sensory ganglia, eliminating recurrence of outbreaks, and/orpreventing viral shedding. The immune system is known to mount a defenseagainst HSV-2, as evidenced by recurrent infections which manifest withfewer, less intense symptoms and decreased frequency over time.

While the ultimate goal of an HSV vaccine would be long-lastingprotection from viral infection, the suppression of disease symptomswould also provide significant health benefits. One of the current goalsfor either a prophylactic or therapeutic vaccine is to reduce clinicalepisodes and viral shedding from primary and latent infections. Threecategories of prophylactic vaccines have been tested in clinical trialswith disappointing results: i) whole virus, ii) protein subunit, andiii) gene-based subunit vaccines (Stanberry et al., Clinical Infect.Dis., 30(3):549-566, 2000). In the 1970s a number of killed virusvaccines were explored, none of which were efficacious. More recently anattenuated HSV was found to be poorly immunogenic. Subunit vaccinesbased on two recombinant glycoproteins have been clinically evaluated incombination with different adjuvant formulations. One developed byChiron contains truncated forms of both glycoprotein D (gD2) andglycoprotein B (gB2) of HSV-2, purified from transfected Chinese HamsterOvary (CHO) cells and formulated with adjuvants alum and MF59. Anotherdeveloped by Glaxo-Smithkline (GSK) contains a truncated gD2 formulatedwith adjuvants alum and 3-O-deacylated monophosphoryl lipid A (MPL).Both vaccines were immunogenic and well tolerated in phase I/II trials.However in phase III analyses, the Chiron vaccine showed no overallefficacy against HSV-2 seroconversion and work was discontinued. The GSKvaccine showed significant efficacy (73-74%) in HSV-1, HSV-2seronegative women volunteers but no efficacy in men.

While even limited vaccine efficacy would beneficially impact HSVsufferers, these trials are testing only a small number of vaccinepossibilities. This is because the vaccine discovery has not beensystematic. Pursuance of a whole-virus vaccine assumes that presentationof the pathogen itself to the immune system will generate optimalimmunity. Indeed the breadth and duration of immune responses to wholepathogen vaccines historically have been better than subunit vaccines.However, pathogenicity of the vaccine strain must be considered. Subunitvaccines, to date, have been selected for vaccine testing based on theirassumed importance in disease pathogenesis and immunogenicity duringinfection. These approaches have identified one candidate against HSVwith limited efficacy in some but no efficacy in other formulations.Thus, new and improved methodologies for herpesvirus vaccine discoveryare needed to protect against herpes diseases.

SUMMARY

Infection and transmission of HSV-2 is a major health concern. Thepresent disclosure provides, inter alia, certain highly effectivevaccines against HSV-2. Such vaccines can be used either therapeuticallyor prophylactically. The present disclosure also provides specificantigens and methods for using the antigens to elicit an immune responseagainst HSV-2.

In one aspect, the present disclosure describes a vaccine formulationcomprising a pharmaceutically-acceptable carrier and at least onepolypeptide consisting of SEQ ID NO: 136 or an immunogenic fragmentthereof, The vaccine formulation may comprise a first polypeptideconsisting of the above SEQ ID NO, a second polypeptide consisting ofSEQ ID NO: 1 or SEQ ID NO: 4 and optionally a third polypeptideconsisting of the other of SEQ ID NOS: 1 and 4. In some embodiments, thesecond or third polypeptide consists of polypeptide fragments of SEQ IDNO: 1, such as the polypeptides of SEQ ID NOS: 2, 8-16, 138 and 139, orimmunogenic fragments thereof. In some embodiments, the vaccineformulation may comprise a first polypeptide consisting of SEQ ID NO:136, a second polypeptide consisting of SEQ ID NO: 4 or SEQ ID NO: 5, athird polypeptide selected from the group consisting of SEQ ID NOS: 2,8-16, 138 and 139, and optionally a fourth polypeptide selected from thegroup consisting of SEQ ID NOS: 2, 8-16, 138 and 139, or immunogenicfragments thereof.

Another aspect of the present invention provides a vaccine formulationcomprising a pharmaceutically acceptable carrier, an adjuvant comprisingone or more purified fractions of Quillaja saponins, and at least onepolypeptide comprising any of SEQ ID NOS: 1, 4, 5, and 136 or animmunogenic fragment thereof. In certain embodiments, at least onepolypeptide comprises one or more polypeptide fragments of SEQ ID NO: 1,such as the polypeptides of SEQ ID NOS: 2, 8-16, 138 and 139.

In still a further aspect, the present invention provides a vaccineformulation where a polypeptide comprising SEQ ID NO: 5 is present inplace of a polypeptide of SEQ ID NO: 4, wherein the polypeptide lacksall or at least an 8 contiguous amino acid residue portion of thetransmembrane domain spanning residues 340-363. The polypeptide may beglycosylated, or may be unglycosylated.

In some embodiments, polypeptides in the vaccine formulations may beconjugated to an immunogenic carrier, for example keyhole limpethemocyanin. In other embodiments, the vaccine formulations furthercomprise an adjuvant. The adjuvant may be one or more purified fractionsof Quillaja saponins, or the adjuvant may comprise a cytokine, or theadjuvant may comprise a cationic peptide with TLR agonist.

The invention provides methods of treating a subject suffering from orsusceptible to HSV-2 infection by administering an effective amount of avaccine formulation disclosed herein. In some embodiments, the methodinhibits HSV-2 symptoms, for example by reducing the number of herpeticlesions, reducing the number of days a subject experiences herpeticlesions, reducing infection by HSV-2 in an uninfected subject,increasing the IgG titer and/or T cell response to one or more HSV-2antigens, and/or reducing the number of herpetic lesions at the onset ofHSV-2 infection.

One aspect of the present invention provides pharmaceutical compositionscomprising two, three, four, or more isolated polypeptides selected frompolypeptides having an amino acid sequence of at least one of SEQ IDNOS: 1-38, 135, 136, 138 and 139, or an immunogenic fragment thereof.

In another aspect, the invention provides vaccine formulations thatinclude a pharmaceutically-acceptable carrier and a polypeptidecomprising at least one of SEQ ID NOS: 1-38, 135, 136, 138 and 139, oran immunogenic fragment thereof. In certain embodiments, the polypeptideconsists of at least one of SEQ ID NOS: 1-38, 135, 136, 138 and 139.

Another aspect of the present invention provides pharmaceuticalcompositions comprising two, three, four, or more isolated nucleic acidshaving a nucleotide sequence that encodes at least one of SEQ ID NOS:1-38, 135, 136, 138 and 139, or an immunogenic fragment thereof. Incertain embodiments, the nucleic acids encode at least one of SEQ IDNOS: 1, 3, 5, 38 or an immunogenic fragment thereof. For example, thenucleic acids may include at least one of SEQ ID NOS: 39-45, 117-129,137, 140 and 141, or a fragment thereof that encodes an immunogenicpolypeptide.

In another aspect, the invention provides vaccine formulations thatinclude a pharmaceutically-acceptable carrier and a nucleic acid havinga nucleotide sequence that encodes at least one of SEQ ID NOS: 1, 3, 5,38, 136 or 138, or an immunogenic fragment thereof. For example, thenucleic acids can have a nucleotide sequence comprising at least one ofSEQ ID NOS: 39, 46, 118, 137 or 140, or a fragment thereof that encodesan immunogenic polypeptide.

Another aspect of the present invention provides a method of inducing animmune response in a subject, comprising administering to said subjectan effective amount of a vaccine formulation or a pharmaceuticalcomposition as described herein.

Yet another aspect of the present invention provides a method ofreducing one or more symptoms of HSV-2 infection in a subject,comprising administering to said subject an effective amount of avaccine formulation or a pharmaceutical composition as described herein.In some embodiments, the symptoms of HSV-2 infection comprise one ormore of lesion formation, pain, irritation, itching, fever, malaise,headache, viral shedding, and prodrome.

A further aspect of the present invention provides a method ofinhibiting the onset of HSV-2 infection, comprising administering aneffective amount of a vaccine formulation or a composition as describedherein.

Applicants disclose another aspect of the present invention, whichprovides a method of inhibiting development of a latent HSV-2 infectionin a subject exposed to HSV-2, comprising administering an effectiveamount of a vaccine formulation or a composition as described herein.

In a related aspect, the present invention provides a method of reducingviral shedding in a subject infected with HSV-2, comprisingadministering an effective amount of a vaccine formulation or acomposition as described herein.

Further, an aspect of the present invention provides a method ofreducing recurrence of outbreaks in a subject infected with HSV-2,comprising administering an effective amount of a vaccine formulation ora composition as described herein.

An additional aspect of the present invention provides a method ofproducing any of the pharmaceutical compositions described above,comprising expressing said two or more polypeptides; and isolating saidtwo or more polypeptides.

Applicants further disclose an aspect of the present invention whichprovides a method for diagnosing severity of symptoms in a subjectedinfected with HSV-2, comprising (i) measuring activation of T cells inresponse to autologous antigen presenting cells (APCs) pulsed with oneor more isolated HSV-2 polypeptides as described herein, and (ii)comparing said levels to reference levels obtained from infectedsubjects experiencing frequent outbreaks; whereby a significant increasein said responses relative to reference levels indicates that saidsubject has less severe symptoms (e.g., the subject is asymptomatic). Asignificant increase in response can, for example, comprise a 1.5-foldor greater, 2-fold or greater, 3-fold or greater, 5-fold or greater,10-fold or greater or even 20-fold or greater increase.

Another aspect of the present invention provides a method for diagnosingseverity of symptoms in a subject infected with HSV-2, comprising (i)measuring activation of T cells from naturally infected or virus-exposedsubjects in response to APCs presenting one or more isolated HSV-2polypeptides selected from polypeptides as described herein, or animmunogenic fragment thereof, and (ii) comparing said levels toreference levels obtained from infected subjects experiencing frequentoutbreaks; whereby a significant decrease in said activation relative toreference levels indicates that said subject has more severe symptoms(e.g., frequent outbreaks).

Another aspect of the present invention provides pharmaceuticalcompositions comprising an antibody that binds to one or more isolatedHSV polypeptides selected from the list as described herein, or animmunogenic fragment thereof.

Moreover, a different aspect of the present invention provides a methodof identifying immunogenic compositions for HSV-2 by testing two, three,four, or more polypeptides selected from polypeptides having an aminoacid sequence as described herein, or an immunogenic fragment thereof,for ability to promote cytokine production in a mammalian T cell,wherein an immunogenic composition is one that elevates levels of acytokine significantly above the levels of that cytokine produced by anaïve mammalian T cell. A significant increase in cytokine levels istypically one that is at least 1.5-fold, 2-fold, 3-fold, 5-fold, 10-foldor even 20-fold the level produced by a naïve cell.

Still another aspect of the present invention provides a method ofdetecting HSV-2 in a sample from a subject, said method comprising (i)contacting said sample with one or more antibodies raised against one ormore polypeptides having an amino acid sequence as described herein oran immunogenic fragment thereof, and (ii) detecting said one or moreantibodies bound to said one or more HSV-2 polypeptide from the sample.

Finally, one aspect of the present invention provides pharmaceuticalcompositions comprising two or more isolated polynucleotides, encodingpolypeptides as described herein, or fragments encoding immunogenicpeptides thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The Figures described below, that together make up the Drawing, are forillustration purposes only, not for limitation.

FIGS. 1A and B depict exemplary graphs illustrating, respectively, CD4⁺and CD8⁺ T cell responses following immunization with gD2 full-lengthprotein, gD2ΔTMR, or gD2 truncated immediately upstream of thetransmembrane domain (denoted 306t).

FIGS. 2A and B depict exemplary graphs illustrating, respectively, CD4⁺and CD8⁺ T cell responses following immunization with pooled,overlapping peptides spanning gL2 or ICP4 fragments encoded by RS1.1,RS1.3.1 and RS1.3.2.

FIGS. 3A and B depict exemplary graphs illustrating, respectively, CD4⁺and CD8⁺ T cell responses following immunization with gD2ΔTMR, orgD2ΔTMR and ICP4.2.

FIGS. 4A and B depict exemplary graphs illustrating the number of IFN-γspot forming units per 2×10⁵ CD4⁺ (Panel A) or CD8⁺ (Panel B) T cells,following immunization with gD2ΔTMR, ICP4.2, gD2ΔTMR plus ICP4, gL2sv.2, UL40 protein, and gL2s v.2 plus UL40 protein.

FIG. 5 illustrates IgG1 and IgG2c antibody titers against gL2s v.2, UL40protein, and gL2s v.2 plus UL40 protein.

FIGS. 6A and B depict exemplary graphs illustrating the average numberof IFN-γ spot forming units per 2×10⁵ CD4⁺ (Panel A) or CD8⁺ (Panel B) Tcells, following immunization with gL2s v.2, ICP4.2, ICP4.2 plus gL2sv.2, ICP4.9, ICP4.9 plus gL2s v.2, ICP4.5, and ICP4.5 plus gL2s v.2.

FIGS. 6C and D depict exemplary graphs illustrating the average numberof IFN-γ spot forming units per 2×10⁵ CD4⁺ (Panel C) or CD8⁺ (Panel D) Tcells, following immunization with gL2s v.2, ICP4.2, ICP4.2 plus gL2sv.2, ICP4.9, ICP4.9 plus gL2s v.2, ICP4.5, and ICP4.5 plus gL2s v.2, asin FIGS. 6A and B, except that APCs were pulsed with pools ofoverlapping peptides spanning the indicated proteins rather than withpurified proteins.

FIG. 7 depicts an exemplary graph illustrating antibody titers againstgL2s v.2, ICP4.5, gL2s v.2 plus ICP4.5, ICP4.9, gL2s v.2 plus ICP4.9,ICP4.2, and gL2s v2 plus ICP4.2.

FIGS. 8A and B depict exemplary graphs illustrating the average numberof IFN-γ spot forming units per 2×10⁵ CD4⁺ (Panel A) or CD8⁺ (Panel B) Tcells, following immunization with pRS1 DNA (encoding ICP4); pUL1 DNA(encoding gL2); pRS1 DNA (encoding ICP4) plus pUL1 DNA (encoding gL2);pUL1 DNA (encoding gL2) with gL2s v.2 protein boost; and gL2s v.2protein.

FIG. 9 depicts an exemplary graph illustrating total IgG antibody titersagainst ICP4.2 and gL2s v.2 (also gD2ΔTMR).

FIGS. 10A and B depict exemplary graphs illustrating the number of IFN-γspot forming units per 2×10⁵ CD4⁺ (Panel A) or CD8⁺ (Panel B) T cells,following immunization with gL2s v.2 protein; pUL1 DNA (encoding gL2);pUL1 DNA (encoding gL2) with gL2s v.2 protein boost; pRS1 DNA (encodingICP4); pRS1 DNA (encoding ICP4) plus pUL1 DNA (encoding gL2); and pUs6DNA (encoding gD2).

FIG. 11 depicts an exemplary graph illustrating the number of IFN-γ spotforming units per 2×10⁵ CD4⁺ (left panel) or CD8⁺ (right panel) T cells,following immunization with pRS1 DNA (encoding ICP4), pRS1.9 DNA(encoding ICP4.9), and pUs4 DNA (encoding gG2) with corresponding DNAboost (first group of mice) or with ICP4.2 protein boost (second groupof mice) on day 21.

FIG. 12 depicts an exemplary graph illustrating the number of IFN-γ spotforming units per 2×10⁵ CD4⁺ (left panel) or CD8⁺ (right panel) T cells,following immunization with pRS1 DNA (encoding ICP4) and pUs4 DNA(encoding gG2) with corresponding DNA boosts on days 21 and 35 (thirdgroup of mice).

DETAILED DESCRIPTION OF THE INVENTION

This application describes vaccines and immunogenic compositions againstHSV-2. Vaccine formulations may include a polypeptide comprising asequence from Table 1 or an immunogenic fragment thereof, or acombination of at least two polypeptides comprising sequences from Table1 or immunogenic fragments thereof. In certain embodiments, thepolypeptide(s) of the vaccines comprise the entire sequence of at leastone of SEQ ID NOS: 1-26, 135, 136, 138 and 139, or consist of the entiresequence of any one of SEQ ID NOS: 1-26, 135, 136, 138 and 139.Immunogenic compositions may include a polypeptide comprising a sequencefrom Table 1 or Table 2 or an immunogenic fragment thereof or acombination of at least two polypeptides comprising sequences from Table1 or Table 2, or immunogenic fragments thereof. In certain embodiments,the polypeptide(s) of the immunogenic compositions comprise the entiresequence of any one of SEQ ID NOS: 1-38, 135, 136, 138 and 139 orconsist of the entire sequence of any one of SEQ ID NO: 1-38, 135, 136,138 and 139. The polypeptides in Tables 1 or 2 may be encoded by SEQ IDNOS: 39-46 and 117-134, 137, 140 and 141 as indicated and/or by cDNAsequences publically available on the world wide web at the hypertextprotocol transfer address of “cbi.nlm.nih.gov/sites/entrez”. cDNA andprotein sequences may also be obtained from any known strains of HSV-2,including HG52, 333, and Strain G. Accordingly, cDNA sequences may beaccessed by gene or protein name from genomic sequence at NC_001798.1,and may be approximately 97% conserved with sequences disclosed atNC_001798.1. As described herein, the polypeptides may be referred to byprotein name, by SEQ ID NO, and/or by the name of the gene encoding theprotein.

The polypeptides can be prepared in a variety of expression systems.Suitable expression systems include E. coli and Baculovirus-basedexpression systems (e.g., in insect cells). Polypeptides prepared usingE. coli are typically full-length and unglycosylated, although truncatedvariants can be prepared. In certain embodiments, these truncatedvariants retain all or part of the signal domain. Polypeptides preparedusing a Baculovirus system typically lack the N-terminal signalsequence, but are fully or partially glycosylated.

In some embodiments, the polypeptides are prepared in non-mammalian cellsystems. When an exogenous signal sequence is used, polypeptides maycontain one or more amino acids at the N-terminal end which correspondto the exogenous signal sequence. An exogenous signal sequence commonlyused in insect expression systems is the honey bee mellitin signalsequence. In other embodiments, the polypeptides may contain one or moreamino acids corresponding to a signal sequence that has been cleaved.Exemplary polypeptides may contain one or more amino acids from amammalian signal sequence that has been left intact or cleaved off,depending on the system used to prepare the polypeptides.

TABLE 1 HSV-2 antigens for vaccines or immunogenic compositions ProteinDNA Gene or Construct SEQ ID SEQ ID Name GenBank Accession No. No.Protein Name GeneID No. Nos. 1 39 RS1 1487291 (duplicated NP_044530.1ICP4 in HSV-2 genome: (duplicated in HSV-2 genome: also 1487290) alsoNP_044544.1) 2 117 RS1.2 1487291 NP_044530.1 ICP4 internal RS1.2corresponds to an amino fragment (ICP4.2) acid sequence of an internalfragment of an RS1 sequence 3 118 UL1 1487292 NP_044470.1 gL2cytoplasmic 4 40 US6ΔTMR 1487358 NP_044536.1 gD2 internal US6ΔTMRcorresponds to deletion (gD2ΔTMR) gD2 with a deletion of amino acids340-363 5 US6 1487358 NP_044536.1 gD2 6 41 RL1 1487287 NP_044529.1ICP34.5 7 42 RL2 1487289 NP_044528.2 ICP0 8 121 RS1.1 1487291NP_044530.1 ICP4 internal RS1.1 corresponds to residues fragment 1-400of RS1 9 122 RS1.3.1 1487291 NP_044530.1 ICP4 internal RS1.3.1corresponds to fragment residues 750-1024 of RS1 10 123 RS1.3.2 1487291NP_044530.1 ICP4 internal RS1.3.2 corresponds to fragment residues1008-1319 of RS1 11 124 RS1.3 1487291 NP_044530.1 ICP4 internal RS1.3corresponds to residues fragment 750-1319_of RS1 12 125 RS1.4 1487291NP_044530.1 ICP4 internal RS1.4 corresponds to residues fragment 340-883of RS1 13 126 RS1.5 1487291 NP_044530.1 ICP4 internal RS1.5 correspondsto residues fragment 775-1318 of RS1 14 127 RS1.6 1487291 NP_044530.1ICP4 internal RS1.6 corresponds to residues fragment 210-1318 of RS1 15128 RS1.7 1487291 NP_044530.1 ICP4 internal RS1.7 has a deletion offragment residues 391-544 of RS1 16 129 RS1.8 1487291 NP_044530.1 ICP4internal RS1.8 has a deletion of fragment residues 786-868 of RS1 17 UL2v.1 1487303 NP_044471.2 uracil DNA glycosylase 135 UL2 v.2 1487303NP_044471.2 uracil DNA glycosylase 18 UL11 1487294 NP_044480.1myristylated tegument protein 19 119 UL1s v.1 1487292 NP_044470.1 gL2secreted 136 137 UL1s v.2 1487292 NP_044470.1 gL2 secreted 20 UL19a1487302 NP_044488.1 VP5 21 120 UL19ΔTEV 1487302 NP_044488.1 VP5 22 UL361487322 NP_044506.1 ICP1/2 23 43 UL36.3.4.1 1487322 NP_044506.1 ICP1/2internal UL 36.3.4.1 corresponds to fragment residues 1318-2280 of UL3624 44 UL36.4.2.5 1487322 NP_044506.1 ICP1/2 internal UL 36.4.2.5corresponds to fragment residues 2253-3122 of UL36 25 UL40 1487327NP_044510.1 ribonucleoside reductase 26 45 US12 1487353 NP_044543.1ICP47 138 140 RS1.9 1487291 NP_044530.1 ICP4 internal RS1.9 has adeletion of fragment residues 391-544 and 786-821 of RS1 139 141 RS1.101487291 NP_044530.1 ICP4 internal RS1.10 has a deletion of fragmentresidues 391-508 and 786-821 of RS1

TABLE 2 Additional HSV-2 antigens for immunogenic compositions ProteinDNA Gene or Construct SEQ ID SEQ ID Name No. No. Protein Name GeneID No.GenBank Accession Nos. 27 134 UL10 1487293 NP_044479.1 gM2 28 UL151487298 NP_044484.1 DNA cleavage/packaging protein 29 UL26.5 1487311NP_044496.1 ICP35 30 UL30 1487316 NP_044500.1 DNA-directed polymerase 31UL5 1487338 NP_044474.1 DNA helicase/primase complex 32 UL8 1487348NP_044477.1 DNA helicase/primase complex 33 UL15.5 1487298 NP_044484.1unknown UL15.5 is an alternate translation of UL15 34 UL32 1487318NP_044502.1 cleavage/packaging protein 35 UL36.4.2 1487322 NP_044506.1ICP1/2 fragment 36 UL54 1487343 NP_044525.1 ICP27 37 133 UL49.5 1487337NP_044520.1 membrane- associated virion protein 38 46 US4 1487356NP_044534.1 gG2Immunogenic HSV-2 Polypeptides

Immunogenic polypeptides or polynucleotides as indicated in Table 1and/or Table 2 may be used in pharmaceutical compositions. The inventionprovides pharmaceutical compositions containing immunogenic polypeptidesor polynucleotides encoding these immunogenic polypeptides together witha pharmaceutical carrier. Antigens from HSV-2 may be identified byscreening immune cells from patients exposed to or infected with HSV-2.Briefly, a library of HSV-2 antigens was expressed by bacteria and mixedwith APCs. The APCs, in turn, processed and presented HSV-2-derivedpeptides to lymphocytes that had been isolated from human patientsexposed to or infected with HSV-2. The patients belonged to severalpopulations: (1) exposed to HSV-2 but seronegative for infection, (2)infected with HSV-2 but asymptomatic, (3) infected with HSV-2 andexperiencing infrequent outbreaks, (4) infected with HSV-2 andexperiencing frequent outbreaks, (5) naïve and (6) seronegative forHSV-2 (HSV-2−) but seropositive for HSV-1 (HSV-1+). Lymphocyte responsesfrom each population were compared for reactivity to HSV-2-derivedpolypeptides, and the screen detected antigens that induced reactivelymphocytes with greater frequency in one patient population as comparedto the others. Infected but asymptomatic, and exposed but seronegativepatients may activate protective immune responses that patients whoexperience frequent outbreaks do not; in particular, exposed butseronegative patients are presumed to have mounted sterilizing immunityto HSV-2 infection. It is believed that a unique set of polypeptideswill activate lymphocytes from these patient populations. Thus, thepresent invention contemplates compositions of the specific HSV-2polypeptides that activate the lymphocytes of infected but asymptomatic,or exposed but seronegative patients or a combination of thesepolypeptides for inhibiting or counteracting infection by HSV-2.

Antigens identified on the basis of their immunogenicity in infected butasymptomatic, or exposed but seronegative patients, are similarlyexpected to be immunogenic in any subject.

In some embodiments, a polypeptide may induce an innate immune response,a humoral immune response, or a cell-mediated immune response. Thecell-mediated immune response may involve CD4+ and/or CD8+ T cells, andin certain embodiments, the immune response involving CD4+ T cells is animmune response in which TH1 cells are activated. In some embodiments,an immunogenic polypeptide avoids induction of TH2 cytokines. In someembodiments, the immune response involving CD4+ T cells is an immuneresponse in which TH17 cells are activated.

Polypeptides (or immunogenic fragments thereof) in compositions of theinvention may induce T cell responses in multiple individuals,regardless of the HLA haplotype of the individuals. Specifically,epitopes in the polypeptides may induce T cell responses in individualswith one or more of the following HLA supertypes: HLA-A2, -A3, -A24,-A1, -B7, -B8, -B27, -B44, -B58, and B62, and HLA-DQB01, -DQB02, -DQB03,-DQB-04, and -DQB05.

In some embodiments, one or more, e.g. two, three, four, or morepolypeptides from Table 1 and/or Table 2 (or immunogenic fragmentsthereof) are provided in a composition of the invention. In someembodiments, two polypeptides from Table 1 and/or Table 2 are providedin a composition of the invention. In other embodiments, threepolypeptides from Table 1 and/or Table 2 are provided in a compositionof the invention. In other embodiments, four polypeptides from Table 1and/or Table 2 are provided in a composition of the invention.

In some embodiments, two, three, four, or more polypeptides from Table 1and/or Table 2 (or immunogenic fragments thereof) are provided togetheras a conjugate. In some embodiments, two polypeptides from Table 1and/or Table 2, or three polypeptides from Table 1 and/or Table 2, orfour polypeptides from Table 1 and/or Table 2, are provided as aconjugate. In some embodiments, two, three, four, or more polypeptidesfrom Table 1 and/or Table 2 are covalently bound to each other, e.g., asa fusion protein. In some embodiments, two polypeptides from Table 1and/or Table 2, or three polypeptides from Table 1 and/or Table 2, orfour polypeptides from Table 1 and/or Table 2, are covalently bound toeach other, e.g. as a fusion protein.

In some embodiments, the compositions comprise two, three, four, or morepolypeptides selected from the group consisting of SEQ ID NOS: 1-38,135, 136, 138 and 139, and may contain or may not contain any otherHSV-2 polypeptides.

In certain embodiments, Applicants provide polypeptides that are atleast 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to apolypeptide encoded by a gene in Table 1 and/or Table 2, or a portion ofsaid polypeptide. In certain embodiments, the homologous polypeptide isat least 8, 10, 15, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 350, 400, 450, or 500 amino acids inlength. In some embodiments, such as those described immediately above,the polypeptide is no more than 300, 350, 400, 450, or 500 amino acidsin length.

An immunogenic composition may also comprise portions of saidpolypeptides and genes, for example deletion mutants, truncationmutants, oligonucleotides, and peptide fragments. In some embodiments,the portions of said proteins are immunogenic.

The immunogenicity of a portion of a protein or a homolog thereof can bereadily determined using the same assays that are used to determine theimmunogenicity of the full-length protein. In some embodiments, theportion of the protein has substantially the same immunogenicity as thefull-length proteins. In some embodiments, the immunogenicity is no morethan 10%, 20%, 30%, 40%, or 50% less than that of the full-lengthprotein. The protein fragments may be, for example, linear, circular, orbranched. In some embodiments, a protein or protein fragment comprisesone or more non-natural amino acids (e.g. an amino acid other than the20 typically found in natural proteins). A non-natural amino acid mayhave an atypical side chain. In addition, peptidomimetics may be used;these may incorporate alterations to the peptide backbone.

Some embodiments of the polypeptide composition described herein includean immunogenic polypeptide that contains a membrane translocatingsequence (MTS), to facilitate introduction of the polypeptide into themammalian cell and subsequent stimulation of the cell-mediated immuneresponse. Exemplary membrane translocating sequences include hydrophobicregion in the signal sequence of Kaposi fibroblast growth factor, theMTS of α-synuclein, β-synuclein, or γ-synuclein, the third helix of theAntennapedia homeodomain, SN50, integrin β3 h-region, HIV Tat, pAntp,PR-39, abaecin, apidaecin, Bac5, Bac7, P. berghei CS protein, and thoseMTSs described in U.S. Pat. Nos. 6,248,558, 6,432,680 and 6,248,558.

In certain embodiments, the immunogenic polypeptide is conjugated (i.e.covalently bound) to another molecule. This may, for example, increasethe half-life, solubility, bioavailability, or immunogenicity of theantigen. Molecules that may be conjugated to an immunogenic polypeptideinclude a carbohydrate, biotin, poly(ethylene glycol) (PEG), polysialicacid, N-propionylated polysialic acid, nucleic acids, polysaccharides,and PLGA. There are many different types of PEG, ranging from molecularweights of below 300 g/mol to over 10,000,000 g/mol. PEG chains can belinear, branched, or with comb or star geometries.

Immunogenic HSV-2 Polypeptides and Nucleic Acids for Use in Vaccines

In certain embodiments, one or more, e.g. two, three, four, or moreimmunogenic fragments or variants thereof are provided in a mixture. Forexample, a vaccine formulation may comprise any one or more of SEQ IDNOS: 1-26, 136, 138 or 139.

In certain embodiments, a vaccine formulation may comprise any one, two,three, or four of ICP4, ICP4.2, ICP4.5, ICP4.9, ICP4.10, gL2, gL2s v.2,gD2ΔTMR and gD2 (SEQ ID NOS: 1-5, 13, 136, 138 and 139), or immunogenicfragment(s) thereof. In certain embodiments, combinations containpolypeptides or immunogenic fragments from only one of ICP4 (SEQ ID NO:1), ICP4.2 (SEQ ID NO: 2), ICP4.5 (SEQ ID NO: 13), ICP4.9 (SEQ ID NO:138) and ICP4.10 (SEQ ID NO: 139). In other embodiments, combinationscontain polypeptides or immunogenic fragments from only one of gD2ΔTMR(SEQ ID NO: 4) and gD2 (SEQ ID NO: 5). In yet other embodiments,combinations contain polypeptides or immunogenic fragments from only oneof gL2 (SEQ ID NO: 3) and gL2s v.2s (SEQ ID NO: 136). In someembodiments, combinations contain polypeptides or immunogenic fragmentsfrom any two of ICP4.2 (SEQ ID NO: 2), ICP4.5 (SEQ ID NO: 13), ICP4.9(SEQ ID NO: 138) and ICP4.10 (SEQ ID NO: 139).

In some embodiments, the vaccine formulation may comprise at least onepolypeptide fragment of SEQ ID NO: 1, such as the polypeptides of SEQ IDNOS: 2, 8-16, 138 and 139. In some embodiments, the vaccine formulationmay comprise at least two polypeptide fragments of SEQ ID NO: 1, such asthe polypeptides of SEQ ID NOS: 2, 8-16, 138 and 139. One or morepolypeptide fragments of SEQ ID NO: 1 may replace SEQ ID NO: 1 in any ofthe vaccine formulations or immunogenic compositions as describedherein.

Exemplary combinations of ICP4, ICP4.2, ICP4.5, ICP4.9, ICP4.10, gL2,gL2s v.2, gD2ΔTMR and gD2 include:

Two antigen combinations ICP4 gL2 or gL2s v.2 SEQ ID NO: 1 SEQ ID NO: 3or SEQ ID NO: 136 ICP4 gD2ΔTMR SEQ ID NO: 1 SEQ ID NO: 4 ICP4 gD2 SEQ IDNO: 1 SEQ ID NO: 5 ICP4.2 gL2 or gL2s v.2 SEQ ID NO: 2 SEQ ID NO: 3 orSEQ ID NO: 136 ICP4.2 gD2ΔTMR SEQ ID NO: 2 SEQ ID NO: 4 ICP4.2 gD2 SEQID NO: 2 SEQ ID NO: 5 gL2 or gL2s v.2 gD2ΔTMR SEQ ID NO: 3 or SEQ ID NO:136 SEQ ID NO: 4 gL2 or gL2s v.2 gD2 SEQ ID NO: 3 or SEQ ID NO: 136 SEQID NO: 5 ICP4.5 gL2 or gL2s v.2 SEQ ID NO: 13 SEQ ID NO: 3 or SEQ ID NO:136 ICP4.5 gD2ΔTMR SEQ ID NO: 13 SEQ ID NO: 4 ICP4.5 gD2 SEQ ID NO: 13SEQ ID NO: 5 ICP4.9 gL2 or gL2s v.2 SEQ ID NO: 138 SEQ ID NO: 3 or SEQID NO: 136 ICP4.9 gD2ΔTMR SEQ ID NO: 138 SEQ ID NO: 4 ICP4.9 gD2 SEQ IDNO: 138 SEQ ID NO: 5 ICP4.10 gL2 or gL2s v.2 SEQ ID NO: 139 SEQ ID NO: 3or SEQ ID NO: 136 ICP4.10 gD2ΔTMR SEQ ID NO: 139 SEQ ID NO: 4 ICP4.10gD2 SEQ ID NO: 139 SEQ ID NO: 5

Three antigen combinations ICP4 gL2 gD2ΔTMR SEQ ID NO: 1 SEQ ID NO: 3SEQ ID NO: 4 ICP4.2 gL2 gD2ΔTMR SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4ICP4.5 gL2 gD2ΔTMR SEQ ID NO: 13 SEQ ID NO: 3 SEQ ID NO: 4 ICP4.9 gL2gD2ΔTMR SEQ ID NO: 138 SEQ ID NO: 3 SEQ ID NO: 4 ICP4.10 gL2 gD2ΔTMR SEQID NO: 139 SEQ ID NO: 3 SEQ ID NO: 4 ICP4 gL2 gD2 SEQ ID NO: 1 SEQ IDNO: 3 SEQ ID NO: 5 ICP4.2 gL2 gD2 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 5ICP4.5 gL2 gD2 SEQ ID NO: 13 SEQ ID NO: 3 SEQ ID NO: 5 ICP4.9 gL2 gD2SEQ ID NO: 138 SEQ ID NO: 3 SEQ ID NO: 5 ICP4.10 gL2 gD2 SEQ ID NO: 139SEQ ID NO: 3 SEQ ID NO: 5 ICP4 gL2s v.2 gD2ΔTMR SEQ ID NO: 1 SEQ ID NO:136 SEQ ID NO: 4 ICP4.2 gL2s v.2 gD2ΔTMR SEQ ID NO: 2 SEQ ID NO: 136 SEQID NO: 4 ICP4.5 gL2s v.2 gD2ΔTMR SEQ ID NO: 13 SEQ ID NO: 136 SEQ ID NO:4 ICP4.9 gL2s v.2 gD2ΔTMR SEQ ID NO: 138 SEQ ID NO: 136 SEQ ID NO: 4ICP4.10 gL2s v.2 gD2ΔTMR SEQ ID NO: 139 SEQ ID NO: 136 SEQ ID NO: 4 ICP4gL2s v.2 gD2 SEQ ID NO: 1 SEQ ID NO: 136 SEQ ID NO: 5 ICP4.2 gL2s v.2gD2 SEQ ID NO: 2 SEQ ID NO: 136 SEQ ID NO: 5 ICP4.5 gL2s v.2 gD2 SEQ IDNO: 13 SEQ ID NO: 136 SEQ ID NO: 5 ICP4.9 gL2s v.2 gD2 SEQ ID NO: 138SEQ ID NO: 136 SEQ ID NO: 5 ICP4.10 gL2s v.2 gD2 SEQ ID NO: 139 SEQ IDNO: 136 SEQ ID NO: 5

Four antigen combinations ICP4.2 ICP4.5 gL2 gD2ΔTMR SEQ ID NO: 2 SEQ IDNO: SEQ ID NO: 3 SEQ ID NO: 4 13 ICP4.2 ICP4.9 gL2 gD2ΔTMR SEQ ID NO: 2SEQ SEQ ID NO: 3 SEQ ID NO: 4 ID NO: 138 ICP4.2 ICP4.10 gL2 gD2ΔTMR SEQID NO: 2 SEQ SEQ ID NO: 3 SEQ ID NO: 4 ID NO: 139 ICP4.2 ICP4.5 gL2 gD2SEQ ID NO: 2 SEQ ID NO: SEQ ID NO: 3 SEQ ID NO: 5 13 ICP4.2 ICP4.9 gL2gD2 SEQ ID NO: 2 SEQ ID NO: SEQ ID NO: 3 SEQ ID NO: 5 138 ICP4.2 ICP4.10gL2 gD2 SEQ ID NO: 2 SEQ ID NO: SEQ ID NO: 3 SEQ ID NO: 5 139 ICP4.2ICP4.5 gL2s v.2 gD2ΔTMR SEQ ID NO: 2 SEQ ID NO: SEQ ID NO: SEQ ID NO: 413 136 ICP4.2 ICP4.9 gL2s v.2 gD2ΔTMR SEQ ID NO: 2 SEQ SEQ ID NO: SEQ IDNO: 4 ID NO: 138 136 ICP4.2 ICP4.10 gL2s v.2 gD2ΔTMR SEQ ID NO: 2 SEQSEQ ID NO: SEQ ID NO: 4 ID NO: 139 136 ICP4.2 ICP4.5 gL2s v.2 gD2 SEQ IDNO: 2 SEQ ID NO: SEQ ID NO: SEQ ID NO: 5 13 136 ICP4.2 ICP4.9 gL2s v.2gD2 SEQ ID NO: 2 SEQ ID NO: SEQ ID NO: SEQ ID NO: 5 138 136 ICP4.2ICP4.10 gL2s v.2 gD2 SEQ ID NO: 2 SEQ ID NO: SEQ ID NO: SEQ ID NO: 5 139136

The individual antigens and combinations described above can alsoinclude additional peptides from or derived from HSV-2, such aspolypeptides comprising sequences selected from SEQ ID NOS: 6-12, 14-26,and SEQ ID NO: 135, or immunogenic fragments thereof.

In some embodiments, the individual antigens and combinations describedabove are provided as isolated nucleic acids. In certain aspects, thenucleic acids have the nucleotide sequence of at least one of SEQ IDNOS: 39-45, 117-129, 137, 140, 141, or an immunogenic fragment thereof.Nucleic acids can be present in compositions of the invention singly orin combinations. Exemplary combinations include nucleic acids encodingfor two or more of ICP4 (SEQ ID NO: 1), ICP4.9 (SEQ ID NO: 138), gL2(SEQ ID NO: 3), gG2 (SEQ ID NO: 38) and gD2 (SEQ ID NO: 5).

ICP4 (SEQ ID NO: 1) Encoded by RS1

RS1 encodes ICP4, a transcriptional transactivator that may interactwith and recruit specific components of the general transcriptionmachinery to viral promoters and stabilize their formation fortranscription initiation. ICP4 contains distinct domains fortransactivation/phosphorylation (approximately spanning amino acidresidues 150-200 of SEQ ID NO: 1), DNA binding (approximately spanningresidues 380-540 of SEQ ID NO: 1), nuclear localization (approximatelyspanning residues 630-730 of SEQ ID NO: 1), and late regulatorytransactivation (approximately spanning residues 1220-1319 of SEQ ID NO:1). The DNA and protein sequence of RS1 may be found by searching forRS1 in the publicly available database, Entrez Gene (on the NCBI NIH website on the World Wide Web, at the hypertext protocol transfer addressof “ncbi.nlm.nih.gov/sites/entrez?db=gene”), in the Human herpesvirus 2complete genome.

In some embodiments, vaccines against HSV-2 include a polypeptidecontaining at least 20 consecutive amino acid residues selected fromresidues 383-766 of ICP4 (SEQ ID NO: 1), but no more than 1000 aminoacids of ICP4 (SEQ ID NO: 1). The polypeptide may also be a variant ofthe at least 20 residue fragment.

In certain embodiments, the polypeptide includes no more than 950, 900,850, 800, 750, 700, 650, 600, 550, 500, 450 or even 400 consecutiveamino acids from ICP4. Exemplary polypeptides correspond approximatelyto amino acids residues of full-length ICP4 as follows: 383-766; 1-400(RS1.1); 750-1024 (RS1.3.1); 1008-1319 (RS1.3.2); 750-1319 (RS1.3);280-785 (RS1.4 comprising the full DNA binding region); 680-1319 (RS1.5comprising the glycosylase/C-terminal region); 208-1319 (RS1.6 which mayalso comprise a Met residue at the N-term end); 1-380 plus 545-1319(RS1.7, in which a region spanning approximately residues 381-544 isdeleted, removing the DNA binding regions); 1-785 plus 870-1319 (RS1.8,in which a region spanning approximately residues 786-869 is deleted,removing the nuclear localization domain), or 1-766, 383-1318, 100-750,400-1300, 250-766, 383-900 of ICP4 (SEQ ID NO: 1) and the like.

ICP4 Internal Fragment ICP4.2 (SEQ ID NO: 2) Encoded by RS1.2

RS1.2 encodes a 391 amino acid fragment denoted ICP4.2.

In specific embodiments, vaccines against HSV-2 include a polypeptidecontaining from 50 to all 391 amino acids residues of ICP4.2 (SEQ ID NO:2), such as from 100 to 391, 200 to 391 or 250 to 350 residues. Inparticular embodiments, the polypeptide includes all of ICP4.2 (SEQ IDNO: 2) or is ICP4.2 (SEQ ID NO: 2) itself. These polypeptides may, forexample, include the full length or fragments of ICP4.2 (SEQ ID NO: 2)described herein with amino acid residues 1-382 or 767-1318 of ICP4 (SEQID NO: 1) or fragments thereof, which, in certain embodiments, areconsecutive with the amino acid residues of ICP4.2 being used. Exemplaryfragments that combine the residues of SEQ ID NO: 2 with select residuesfrom 1-382 or 767-1318 of SEQ ID NO: 1 are described above.

An immunogenic fragment of ICP4.2 comprises at least one immunogenicportion, as measured experimentally or identified by algorithm. Peptidesidentified by such methods include the following:

(SEQ ID NO: 47) GLAHVAAAV (SEQ ID NO: 48) FISGSVARA (SEQ ID NO: 49)QYALITRLL (SEQ ID NO: 50) RYDRAQKGF (SEQ ID NO: 51) GYAMAAGRF(SEQ ID NO: 52) PPHADAPRL (SEQ ID NO: 53) KPAAAAAPL (SEQ ID NO: 54)SEAAVAAV (SEQ ID NO: 55) FGWGLAHV (SEQ ID NO: 56) YALITRLLY(SEQ ID NO: 57) ALPRSPRLL (SEQ ID NO: 58) DLLFQNQSL (SEQ ID NO: 59)ADLLFQNQS (SEQ ID NO: 60) ARNSSSFIS (SEQ ID NO: 61) QACFRISGA(SEQ ID NO: 62) FVRDALVLM (SEQ ID NO: 63) FDGDLAAVP (SEQ ID NO: 64)GLGDSRPGL (SEQ ID NO: 65) WAPELGDAA (SEQ ID NO: 66) ECLAACRGI(SEQ ID NO: 67) RAWLRELRF.

Thus, in some aspects, this application provides an immunogenic fragmentof ICP4.2. The fragments, in some instances, are close in size to thefull-length polypeptide. For example, they may lack at most one, two,three, four, five, ten, or twenty amino acids from one or both termini.In other embodiments, the fragment is 100-391 amino acids in length, or150-391, or 200-391, or 250-391 amino acids in length. Other exemplaryfragments are amino acid residues 1-350, 1-300, 1-250, 1-200, 1-150,1-100, 1-50, 50-391, 50-350, 50-300, 50-250, 50-200, 50-150, 50-100,100-391, 100-350, 100-300, 100-250, 100-200, 100-150, 150-391, 150-350,150-300, 150-250, 150-200, 200-391, 200-350, 200-300, 200-250, 250-391,250-350, 250-300, 300-391 and 350-391. The fragments described above orsub-fragments thereof (e.g., fragments of 8-50, 8-30, or 8-20 amino acidresidues) preferably have one of the biological activities describedbelow, such as increasing the T cell response by at least 1.5 fold or 2fold. A fragment may be used as the polypeptide in the vaccinesdescribed herein or may be fused to another protein, protein fragment ora polypeptide.

In certain aspects, this application provides immunogenic polypeptideswith at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to ICP4.2 or animmunogenic fragment thereof.

Glycoprotein L-2 (SEQ ID NO: 3 or SEQ ID NO: 136) Encoded by UL1

UL1 encodes Glycoprotein L-2 (gL2), a heterodimer glycoprotein that isrequired for the fusion of viral and cellular membranes and enables thevirus to enter the host cell. The DNA and protein sequence of UL1 may befound by searching in the publicly available database, Entrez Gene (onthe NCBI NIH web site on the World Wide Web, at the hypertext protocoltransfer address of “ncbi.nlm.nih.gov/sites/entrez?db=gene”), in theHuman herpesvirus 2 complete genome.

In some embodiments, the polypeptide may be a cytoplasmic form of UL1(SEQ ID NO:3). In other embodiments, the polypeptide may be a secretedform of UL1, which lacks one or more amino acids of the signal sequence.An exemplary polypeptide of the secreted form of UL1 is the polypeptideof SEQ ID NO: 136. In certain embodiments, this polypeptide will notform an aggregate after it is substantially purified. In someembodiments, the polypeptide will contain one or more amino acidscorresponding to a signal sequence that has been cleaved. The signalsequence may be a mammalian signal sequence or may be a non-mammaliansignal sequence, depending on the system from which the polypeptide waspurified.

In some embodiments, vaccines against HSV-2 include a polypeptidecontaining at least 20 consecutive amino acid residues selected fromresidues 1-224 or 1-200 of gL2 (SEQ ID NO: 3 or SEQ ID NO: 136), but nomore than 224 or 200 amino acids of gL2 (SEQ ID NO: 3 or SEQ ID NO:136). The polypeptide may also be a variant of the at least 20 residuefragment.

In some embodiments, the polypeptide is at least 85% identical to afragment of 150-200 or 200-250 amino acids of SEQ ID NO: 3 or SEQ ID NO:136.

In certain embodiments, the polypeptide includes no more than 200 or 100consecutive amino acids from gL2. Exemplary polypeptides are amino acidsresidues 1-20, 21-40, 41-60, of 61-80, 81-100, 101-120, 121-140,141-160, 161-180, 181-200, 201-221 of gL2 (SEQ ID NO: 3 or amino acidsresidues 1-20, 21-40, 41-60, of 61-80, 81-100, 101-120, 121-140,141-160, 161-180, or 181-200 SEQ ID NO: 136) and the like.

In other aspects, this application provides an immunogenic fragment ofgL2. An immunogenic fragment of gL2 comprises at least one immunogenicportion, as measured experimentally or identified by algorithm. Peptidesidentified by such methods include the following:

(SEQ ID NO: 100) AYLVNPFLF (SEQ ID NO: 101) PFLFAAGFL (SEQ ID NO: 102)TEYVLRSVI (SEQ ID NO: 103) GSQATEYVL (SEQ ID NO: 104) RIDGIFLRY(SEQ ID NO: 105) FLEDLSHSV (SEQ ID NO: 106) YVLRSVIAK (SEQ ID NO: 107)YVLRSVIAK (SEQ ID NO: 108) AYLVNPFLF (SEQ ID NO: 109) ETTTRRALY(SEQ ID NO: 110) RIDGIFLRY (SEQ ID NO: 111) YLVNPFLFA (SEQ ID NO: 112)FVCLFGLVV (SEQ ID NO: 113) LYKEIRDAL (SEQ ID NO: 114) GLDTFLWDR(SEQ ID NO: 115) RVSPTRGRR (SEQ ID NO: 115) YVLRSVIAK (SEQ ID NO: 116)GLDTFLWDR (SEQ ID NO: 117) DILRVPCMR (SEQ ID NO: 118) DRHAQRAYLGlycoprotein D-2 (SEQ ID NO: 5) Encoded by US6 and Internally-DeletedGlycoprotein D-2 (SEQ ID NO: 4) Encoded by US6ΔTMR

US6 encodes envelope glycoprotein D-2 (gD2), an envelope glycoproteinthat binds to host cell entry receptors and may trigger fusion of thevirus with the host membrane. The gD2 protein has several distinctdomains, including a signal domain (amino acid residues 1-25) which iscleaved from the mature protein, and a transmembrane domain (spanningapproximately amino acids residues 340-363). The DNA and proteinsequence of US6 may be found by searching in the publicly availabledatabase, Entrez Gene (on the NCBI NIH web site on the World Wide Web,at the hypertext protocol transfer address of“ncbi.nlm.nih.gov/sites/entrez?db=gene”), in the Human herpesvirus 2complete genome.

In some embodiments, vaccines against HSV-2 include a polypeptidecomprising gD2 that is missing all or part of the transmembrane domain(which spans approximately amino acids residues 340-363 inclusive) aswell as the signal sequence. In other embodiments, the deleted regionmay additionally include 5-10 amino acids of the sequence flanking thetransmembrane domain. The deleted region may also comprise a portion ofthe transmembrane domain, for example at least 3 amino acids betweenresidues 340-363. In some embodiments, at least one residue in thetransmembrane domain has been modified, deleted or substituted, suchthat the transmembrane domain is no longer functional. For example, avariant may have its internal deletion begin at amino acid residue 336,337, 338, 339, 340, 341, 342, 343, 344, 345 or 346 and end at amino acidresidue 358, 359, 360, 361, 362, 363, 364, 365, 366, 367 or 368.

A construct encoding gD2 which is missing amino acid residues 340-363(the transmembrane domain) is called US6ΔTMR (SEQ ID NO: 40). Thecorresponding protein is denoted gD2ΔTMR (SEQ ID NO: 4). In otherembodiments, an immunogenic fragment of gD2 or gD2ΔTMR may comprise adeletion in a portion of the transmembrane domain, and/or may comprise adeletion in the flanking sequence outside of the transmembrane domain.

In other aspects, this application provides an immunogenic fragment ofgD2 or gD2ΔTMR. An immunogenic fragment of gD2 or gD2ΔTMR comprises atleast one immunogenic portion, as measured experimentally or identifiedby algorithm. Peptides identified by such methods include the following:

(SEQ ID NO: 68) ALAGSTLAV (SEQ ID NO: 69) LLEDPAGTV (SEQ ID NO: 70)VIGGIAFWV (SEQ ID NO: 71) TVYYAVLER (SEQ ID NO: 72) KYALADPSL(SEQ ID NO: 73) AFETAGTYL (SEQ ID NO: 74) APSNPGLII (SEQ ID NO: 75)IPITVYYAV (SEQ ID NO: 76) APPSHQPLF (SEQ ID NO: 77) FLMHAPAFE(SEQ ID NO: 78) FSAVSEDNL (SEQ ID NO: 79) VYYAVLER (SEQ ID NO: 80)IGMLPRFI (SEQ ID NO: 81) YTECPYNKS (SEQ ID NO: 82) FLMHAPAFE(SEQ ID NO: 83) NLGFLMHAP (SEQ ID NO: 84) VIGGIAFWV (SEQ ID NO: 85)GIAFWVRRR (SEQ ID NO: 86) SEDNLGFLM (SEQ ID NO: 87) RTQPRWSYY(SEQ ID NO: 88) IAFWVRRRA (SEQ ID NO: 89) LVIGGIAFW (SEQ ID NO: 90)FWVRRRAQM (SEQ ID NO: 91) PYTSTLLPP (SEQ ID NO: 92) VGTAALLVV(SEQ ID NO: 93) TAALLVVAV (SEQ ID NO: 94) TSTLLPPEL (SEQ ID NO: 95)GTVSSQIPP (SEQ ID NO: 96) TAGTYLRLV (SEQ ID NO: 97) GVTVDSIGM(SEQ ID NO: 98) AFWVRRRAQ (SEQ ID NO: 99) RVYHIQPSL

Thus, in some aspects, this application provides an immunogenic fragmentof gD2 (SEQ ID NO: 5) or gD2ΔTMR (SEQ ID NO: 4). The fragments, in someinstances, are close in size to the full-length polypeptide. Forexample, they may lack at most one, two, three, four, five, ten, ortwenty amino acids from one or both termini. In other embodiments, thefragment is 100-393 amino acids in length, or 150-393, or 200-393, or250-393 amino acids in length. Other exemplary fragments are amino acidresidues 1-350, 1-300, 1-250, 1-200, 1-150, 1-100, 1-50, 50-393, 50-350,50-300, 50-250, 50-200, 50-150, 50-100, 100-393, 100-350, 100-300,100-250, 100-200, 100-150, 150-393, 150-350, 150-300, 150-250, 150-200,200-393, 200-350, 200-300, 200-250, 250-393, 250-350, 250-300, 300-393and 350-393. The fragments described above or sub-fragments thereof(e.g., fragments of 8-50, 8-30, or 8-20 amino acid residues) preferablyhave one of the biological activities described below, such asincreasing the T cell response by at least 1.5 fold or 2 fold. Afragment may be used as the polypeptide in the vaccines described hereinor may be fused to another protein, protein fragment or a polypeptide.

In other embodiments, the polypeptide comprises the entire sequence ofSEQ ID NO: 4 or SEQ ID NO: 5, or consists of the entire sequence of SEQID NO: 4 or SEQ ID NO: 5. In certain embodiments, an immunogenicfragment of gD2 retains all or part of the signal domain (amino acidresidues 1-25) and/or the transmembrane domain (amino acids residues340-363).

In certain embodiments, polypeptides have less than 20%, 30%, 40%, 50%,60% or 70% homology with human autoantigens. Examples of suchautoantigens include UL6 from HSV-1 and gK or UL53 from HSV-2.

In certain aspects, this application provides immunogenic polypeptideswith at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to gD2ΔTMR, oran immunogenic fragment thereof.

ICP4 Internal Fragment ICP4.5 (SEQ ID NO: 13) Encoded by RS1.5

RS1.5 encodes a 544 amino acid fragment corresponding to residues775-1318 of ICP4, denoted ICP4.5. The DNA and protein sequences of RS1.5may be found by searching for RS1 in the publicly available database,Entrez Gene (on the NCBI NIH web site on the World Wide Web, at thehypertext protocol transfer address of“ncbi.nlm.nih.gov/sites/entrez?db=gene”), in the Human herpes virus 2complete genome.

In specific embodiments, vaccines against HSV-2 include a polypeptidecontaining from 50 to all 544 amino acids residues of ICP4.5 (SEQ ID NO:13), such as 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 544residues. In particular embodiments, the polypeptide includes all ofICP4.5 (SEQ ID NO: 13) or is ICP4.5 (SEQ ID NO: 13) itself. Thesepolypeptides may, for example, include the full length or fragments ofICP4.5 (SEQ ID NO: 13) described herein with amino acid residues 1-774of ICP4 (SEQ ID NO: 1) or fragments thereof, which, in certainembodiments, are consecutive with the amino acid residues of ICP4.5being used. Exemplary fragments that combine the residues of SEQ ID NO:13 with select residues from 1-774 of SEQ ID NO: 1 are described above.

An immunogenic fragment of ICP4.5 comprises at least one immunogenicportion, as measured experimentally or identified by algorithm. Thus, insome aspects, this application provides an immunogenic fragment ofICP4.5. The fragments, in some instances, are close in size to thefull-length polypeptide. For example, they may lack at most one, two,three, four, five, ten, or twenty amino acids from one or both termini.In other embodiments, the fragment is 50-544 amino acids in length, or100-544, or 150-544, or 200-544, or 250-544, or 300-544, or 350-544, or400-544, or 450-544, or 500-544 amino acids in length. Other exemplaryfragments are amino acid residues 1-500, 1-450, 1-400, 1-350, 1-300,1-250, 1-200, 1-150, 1-100, 1-50, 50-544, 50-500, 50-450, 50-400,50-350, 50-300, 50-250, 50-200, 50-150, 50-100, 100-544, 100-500,100-450, 100-400, 100-350, 100-300, 100-250, 100-200, 100-150, 150-544,150-500, 150-450, 150-400, 150-350, 150-300, 150-250, 150-200, 200-544,200-500, 200-450, 200-400, 200-350, 200-300, 200-250, and so forth. Thefragments described above or sub-fragments thereof (e.g., fragments of8-50, 8-30, or 8-20 amino acid residues) preferably have one of thebiological activities described below, such as increasing the T cellresponse by at least 1.5 fold or 2 fold. A fragment may be used as thepolypeptide in the vaccines described herein or may be fused to anotherprotein, protein fragment or a polypeptide.

In certain aspects, this application provides immunogenic polypeptideswith at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to ICP4.5 or animmunogenic fragment thereof.

ICP4 Fragment ICP4.9 (SEQ ID NO: 138) Encoded by RS1.9, and ICP4Fragment ICP4.10 (SEQ ID NO: 139) Encoded by RS1.10

RS1.9 encodes a 1130 amino acid fragment of ICP4, carrying a doubleinternal deletion of residues 391-544 and residues 786-821 of ICP4,denoted ICP4.9. RS1.10 encodes a 1166 amino acid fragment of ICP4,carrying a double internal deletion of residues 391-508 and residues786-821 of ICP4, denoted ICP4.10. The DNA and protein sequences of RS1.9and RS1.10 may be found by searching for RS1 in the publicly availabledatabase, Entrez Gene (on the NCBI NIH web site on the World Wide Web,at the hypertext protocol transfer address of“ncbi.nlm.nih.gov/sites/entrez?db=gene”), in the Human herpesvirus 2complete genome.

In specific embodiments, vaccines against HSV-2 include a polypeptidecontaining from 50 to all 1130 or 1166 amino acids residues of ICP4.9(SEQ ID NO: 138) or ICP4.10 (SEQ ID NO: 139), such as 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1130 or 1166 residues. In particular embodiments,the polypeptide includes all of ICP4.9 (SEQ ID NO: 138) or ICP4.10 (SEQID NO: 139), or is ICP4.9 (SEQ ID NO: 138) or ICP4.10 (SEQ ID NO: 139)itself. These polypeptides may, for example, include the full length orfragments of ICP4.9 (SEQ ID NO: 138) or ICP4.10 (SEQ ID NO: 139)described herein.

An immunogenic fragment of ICP4.9 or ICP4.10 comprises at least oneimmunogenic portion, as measured experimentally or identified byalgorithm. Thus, in some aspects, this application provides animmunogenic fragment of ICP4.9 or ICP4.10. The fragments, in someinstances, are close in size to the full-length polypeptide. Forexample, they may lack at most one, two, three, four, five, ten, ortwenty amino acids from one or both termini. In other embodiments, thefragment is 50-1130 amino acids in length, or 100-1130, 150-1130, or200-1130, or 250-1130, or 300-1130, or 400-1130, or 500-1130, or600-1130, or 700-1130, or 800-1130, or 900-1130, or 1000-1130 aminoacids in length. Other exemplary fragments are amino acid residues1-1130, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200,1-150, 1-100, 1-50, 50-1130, 50-1000, 50-900, 50-800, 50-700, 50-600,50-500, 50-400, 50-300, 50-250, 50-200, 50-150, 50-100, 100-1130,100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300,100-250, 100-200, 100-150, and so forth. The fragments described aboveor sub-fragments thereof (e.g., fragments of 8-50, 8-30, or 8-20 aminoacid residues) preferably have one of the biological activitiesdescribed below, such as increasing the T cell response by at least 1.5fold or 2 fold. A fragment may be used as the polypeptide in thevaccines described herein or may be fused to another protein, proteinfragment or a polypeptide.

In certain embodiments, an analog of ICP4.9 is based on SEQ ID NO: 1,where at least 50, 75, 100, 125, 130, 140, 145 or 150 residues fromresidues 391-544 are deleted. Separately or in combination, at least 20,25, or 30 residues from residues 786-821 are deleted.

In certain embodiments, an analog of ICP4.10 is based on SEQ ID NO: 1,where at least 25, 50, 75, 90, 95, 100, 105, 110 or 115 residues fromresidues 391-508 are deleted. Separately or in combination, at least 25,50, 60, 65, 70 or 75 residues from residues 786-821 are deleted.

In certain aspects, this application provides immunogenic polypeptideswith at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to ICP4.9 orICP4.10, or an immunogenic fragment or analog thereof.

Additional Features of HSV-2 Polypeptides

Typically, the polypeptides present in the vaccine formulations orpharmaceutical compositions described herein are immunogenic, eitheralone or as a variant, which includes polypeptides fused to anotherpolypeptide or mixed with or complexed to an adjuvant. Variants alsoinclude sequences with less than 100% sequence identity, as describedherein. In addition, one may use fragments, precursors and analogs thathave an appropriate immunogenicity.

These polypeptides may be immunogenic in mammals, for example, mice,guinea pigs, or humans. An immunogenic polypeptide is typically onecapable of raising a significant immune response in an assay or in asubject. Alternatively, an immunogenic polypeptide may (i) induceproduction of antibodies, e.g., neutralizing antibodies, that bind tothe polypeptide (ii) induce T_(H)1 immunity, (iii) activate the CD8⁺ Tcell response, for example by increasing the number of CD8⁺ T cells,increasing localization of CD8⁺ T cells to the site of infection orreinfection, (iv) induce T_(H)17 immunity, and/or (v) activate innateimmunity. In some embodiments, an immunogenic polypeptide causes theproduction of a detectable amount of antibody specific to that antigen.

In certain embodiments, polypeptides have less than 20%, 30%, 40%, 50%,60% or 70% homology with human autoantigens.

A polypeptide may comprise one or more immunogenic portions and one ormore non-immunogenic portions. The immunogenic portions may beidentified by various methods, including protein microarrays,ELISPOT/ELISA techniques, and/or specific assays on different deletionmutants (e.g., fragments) of the polypeptide in question. Immunogenicportions may also be identified by computer algorithms. Some suchalgorithms, like EpiMatrix (produced by EpiVax), use a computationalmatrix approach. Other computational tools for identifying antigenicepitopes include PEPVAC (Promiscuous EPitope-based VACcine, hosted byDana Farber Cancer Institute on the world wide web at the hypertextprotocol transfer address of “immunax.dfci.harvard.edu/PEPVAC”), MHCPred(which uses a partial least squares approach and is hosted by The JennerInstitute on the world wide web at the hypertext protocol transferaddress of “jenner.ac.uk/MHCPred”), and Syfpeithi, hosted on the worldwide web at the hypertext protocol transfer address of “syfpeithi.de/”.

In some embodiments, the vaccine or pharmaceutical composition maycomprise fusion proteins and/or fusion DNA constructs. The underlyingDNA sequences above may be modified in ways that do not affect thesequence of the protein product. For instance, the DNA sequence may becodon-optimized to improve expression in a host such as E. coli or aninsect cell line (e.g., using the baculovirus expression system) ormammalian (e.g., Chinese Hamster Ovary) cell line. In certainembodiments, the DNA sequence may comprise an exogenous sequence, suchas an exogenous signal sequence, for expression in non-mammalian cells.In particular embodiments, such as when smaller related polypeptides,including those having a molecular weight less than about 5000 daltons,e.g., 1500 to 5000 daltons, are used, modification may be useful ineliciting the desired immune response. For example, the smallerpolypeptides can be conjugated to an appropriate immunogenic carriersuch as proteins from other pathogenic organisms or viruses (e.g.,tetanus toxoid), large proteins (e.g., keyhole limpet hemocyanin) or thelike. Conjugation may be direct or indirect (e.g., via a linker). Inother particular embodiments, a fusion protein may comprise apolypeptide disclosed above or an immunogenic fragment or variantthereof and a tag. A tag may be N-terminal or C-terminal. For instance,tags may be added to the nucleic acid or polypeptide to facilitatepurification, detection, solubility, or confer other desirablecharacteristics on the protein or nucleic acid. For instance, apurification tag may be a peptide, oligopeptide, or polypeptide that maybe used in affinity purification. Examples include His, GST, TAP, FLAG,myc, HA, MBP, VSV-G, thioredoxin, V5, avidin, streptavidin, BCCP,Calmodulin, Nus, S tags, lipoprotein D, and 13 galactosidase. In someembodiments, the fused portion is short. Thus, in some instances, thefusion protein comprises no more than 1, 2, 3, 4, 5, 10, 20, or 50additional amino acids on one or both termini of a polypeptide describedabove, such as consecutive amino acids from any of the polypeptides inTable 1.

In some embodiments, tags, secretion signals, or other signal sequencesmay be added to the C-terminal end and/or to the N-terminal end of thepolypeptide. Tags may be used to aid in purification of expressedpolypeptides. Exemplary tags include HHHHHH (SEQ ID NO: 130) andMSYYHHHHHH (SEQ ID NO: 131). Secretion signals may be optimized for usewith non-mammalian cells, such as insect cells. An exemplary secretionsignal is MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 132).

A detection tag may be used to detect the tag and, consequently, anyamino acid sequence fused to it. Detection tags include fluorescentproteins, proteins that bind a fluorescent label, and proteins that bindan electron-dense moeity. Examples of fluorescent proteins includedsRed, mRFP, YFP, GFP, CFP, BFP, and Venus. An example of a protein thatbinds a fluorescent or electron-dense label is FlAsH.

Another aspect disclosed herein is an antibody preparation generatedagainst a composition of the invention (e.g., a composition comprisingone or more, or two or more of the polypeptides listed in Table 1). Anyof a variety of antibodies are included. Such antibodies include, e.g.,polyclonal, monoclonal, recombinant, humanized or partially humanized,single chain, Fab, and fragments thereof, etc. The antibodies can be ofany isotype, e.g., IgA, IgG, various IgG isotypes such as IgG₁, IgG₂,IgG_(2a), IgG_(2b), IgG₃, IgG₄, etc.; and they can be from any animalspecies that produces antibodies, including goat, rabbit, mouse, chickenor the like. In some embodiments, Fab molecules are expressed andassembled in a genetically transformed host like E. coli. A lambdavector system is available thus to express a population of Fab′ s with apotential diversity equal to or exceeding that of the subject generatingthe predecessor antibody. See Huse et al. (1989), Science 246, 1275-81.

Components of Vaccines and Pharmaceutical Compositions

In certain embodiments, the vaccines and pharmaceutical compositionscomprise one or more of the polypeptides and nucleic acids describedabove and one or more of the following: an adjuvant, stabilizer, buffer,surfactant, controlled-release component, salt, preservative, and anantibody specific to said antigen.

Adjuvants

The vaccine formulations and pharmaceutical compositions describedherein may each include an adjuvant. Adjuvants can be broadly separatedinto two classes, based on their principal mechanisms of action: vaccinedelivery systems and immunostimulatory adjuvants (see, e.g., Singh etal., Curr. HIV Res. 1:309-20, 2003). Vaccine delivery systems are oftenparticulate formulations, e.g., emulsions, microparticles,immune-stimulating complexes (ISCOMs), which may be, for example,particles and/or matrices, and liposomes. In contrast, immunostimulatoryadjuvants are sometimes derived from pathogens and can representpathogen associated molecular patterns (PAMP), e.g., lipopolysaccharides(LPS), monophosphoryl lipid (MPL), or CpG-containing DNA, which activatecells of the innate immune system.

Alternatively, adjuvants may be classified as organic and inorganic.Inorganic adjuvants include aluminum salts such as aluminum phosphate,amorphous aluminum hydroxyphosphate sulfate, and aluminum hydroxide,which are commonly used in human vaccines. Organic adjuvants compriseorganic molecules including macromolecules. An example of an organicadjuvant is cholera toxin.

Adjuvants may also be classified by the response they induce, andadjuvants can activate more than one type of response. In someembodiments, the adjuvant induces the activation of CD4⁺ T cells. Theadjuvant may induce activation of T_(H)1 cells and/or activation ofT_(H)17 cells and/or activation of T_(H)2 cells. Alternately, theadjuvant may induce activation of T_(H)1 cells and/or T_(H)17 cells butnot activation of T_(H)2 cells, or vice versa. In some embodiments, theadjuvant induces activation of CD8⁺T cells. In further embodiments, theadjuvant may induce activation of Natural Killer T (NKT) cells. In someembodiments, the adjuvant induces the activation of T_(H)1 cells orT_(H)17 cells or T_(H)2 cells. In other embodiments, the adjuvantinduces the activation of B cells. In yet other embodiments, theadjuvant induces the activation of APCs. These categories are notmutually exclusive; in some cases, an adjuvant activates more than onetype of cell.

In certain embodiments, an adjuvant is a substance that increases thenumbers or activity of APCs such as dendritic cells. In certainembodiments, an adjuvant promotes the maturation of APCs such asdendritic cells. In some embodiments, the adjuvant is or comprises asaponin. Typically, the saponin is a triterpene glycoside, such as thoseisolated from the bark of the Quillaja saponaria tree. A saponin extractfrom a biological source can be further fractionated (e.g., bychromatography) to isolate the portions of the extract with the bestadjuvant activity and with acceptable toxicity. Typical fractions ofextract from Quillaja saponaria tree used as adjuvants are known asfractions A and C. An exemplary saponin adjuvant is QS-21 (fraction C),which is available from Antigenics. QS-21 is anoligosaccharide-conjugated small molecule. Optionally, QS-21 may beadmixed with a lipid such as 3D-MPL or cholesterol.

A particular form of saponins that may be used in vaccine formulationsdescribed herein is immunostimulating complexes (ISCOMs). ISCOMs are anart-recognized class of adjuvants, that generally comprise Quillajasaponin fractions and lipids (e.g., cholesterol and phospholipids suchas phosphatidyl choline). In certain embodiments, an ISCOM is assembledtogether with a polypeptide or nucleic acid of interest. However,different saponin fractions may be used in different ratios. Inaddition, the different saponin fractions may either exist together inthe same particles or have substantially only one fraction per particle(such that the indicated ratio of fractions A and C are generated bymixing together particles with the different fractions). In thiscontext, “substantially” refers to less than 20%, 15%, 10%, 5%, 4%, 3%,2% or even 1%. Such adjuvants may comprise fraction A and fraction Cmixed into a ratio of 70-95 A:30-5 C, such as 70 A:30 C to 75 A:25 C; 75A:25 C to 80 A:20 C; 80 A:20 C to 85 A:15 C; 85 A:15 C to 90 A:10 C; 90A:10 C to 95 A:5 C; or 95 A:5 C to 99 A:1 C. ISCOMatrix, produced byCSL, and AbISCO 100 and 300, produced by Isconova, are ISCOM matricescomprising saponin, cholesterol and phospholipid (lipids from cellmembranes), which form cage-like structures typically 40-50 nm indiameter. Posintro, produced by Nordic Vaccines, is an ISCOM matrixwhere the immunogen is bound to the particle by a multitude of differentmechanisms, e.g., electrostatic interaction by charge modification,incorporation of chelating groups, or direct binding.

In some embodiments, the adjuvant is a TLR ligand. TLRs are proteinsthat may be found on leukocyte membranes, and recognize foreign antigens(including microbial antigens). An exemplary TLR ligand is IC-31, whichis available from Intercell. IC-31 comprises an anti-microbial peptide,KLK, and an immunostimulatory oligodeoxynucleotide, ODN1a. IC-31 hasTLR9 agonist activity. Another example is CpG-containing DNA. Differentvarieties of CpG-containing DNA are available from Prizer (Coley):VaxImmune is CpG 7909 (a (CpG)-containing oligodeoxy-nucleotide), andActilon is CpG 10101 (a (CpG)-containing oligodeoxy-nucleotide).

In some embodiments, the adjuvant is a nanoemulsion. One exemplarynanoemulsion adjuvant is Nanostat Vaccine, produced by Nanobio. Thisnanoemulsion is a high-energy, oil-in-water emulsion. This nanoemulsiontypically has a size of 150-400 nanometers, and includes surfactants toprovide stability. More information about Nanostat can be found in U.S.Pat. Nos. 6,015,832, 6,506,803, 6,559,189, 6,635,676, and 7,314,624.

In some embodiments, an adjuvant includes a cytokine. In someembodiments, the cytokine is an interleukin such as IL-1, IL-6, IL-12,IL-17 and IL-23. In some embodiments, the cytokine isgranulocyte-macrophage colony-stimulating factor (GM-CSF). The adjuvantmay include cytokine as a purified polypeptide. Alternatively, theadjuvant may include nucleic acids encoding the cytokine.

Adjuvants may be covalently bound to antigens (e.g., the polypeptidesdescribed above). In some embodiments, the adjuvant may be a proteinwhich induces inflammatory responses through activation of APCs. In someembodiments, one or more of these proteins can be recombinantly fusedwith an antigen of choice, such that the resultant fusion moleculepromotes dendritic cell maturation, activates dendritic cells to producecytokines and chemokines, and ultimately, enhances presentation of theantigen to T cells and initiation of T cell responses (see Wu et al.,Cancer Res 2005; 65(11), pp 4947-4954). Other exemplary adjuvants thatmay be covalently bound to antigens comprise polysaccharides, syntheticpeptides, lipopeptides, and nucleic acids.

The adjuvant can be used alone or in combination of two or more kindsAdjuvants may be directly conjugated to antigens. Adjuvants may also becombined to increase the magnitude of the immune response to theantigen. Typically, the same adjuvant or mixture of adjuvants is presentin each dose of a vaccine. Optionally, however, an adjuvant may beadministered with the first dose of vaccine and not with subsequentdoses (i.e. booster shots). Alternatively, a strong adjuvant may beadministered with the first dose of vaccine and a weaker adjuvant orlower dose of the strong adjuvant may be administered with subsequentdoses. The adjuvant can be administered before the administration of theantigen, concurrent with the administration of the antigen or after theadministration of the antigen to a subject (sometimes within 1, 2, 6, or12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants areappropriate for human patients, non-human animals, or both.

Additional Components of Vaccines and Pharmaceutical Compositions

In addition to the antigens and the adjuvants described above, a vaccineformulation or pharmaceutical composition may include one or moreadditional components.

In certain embodiments, the vaccine formulation or pharmaceuticalcomposition may include one or more stabilizers such as sugars (such assucrose, glucose, or fructose), phosphate (such as sodium phosphatedibasic, potassium phosphate monobasic, dibasic potassium phosphate, ormonosodium phosphate), glutamate (such as monosodium L-glutamate),gelatin (such as processed gelatin, hydrolyzed gelatin, or porcinegelatin), amino acids (such as arginine, asparagine, histidine,L-histidine, alanine, valine, leucine, isoleucine, serine, threonine,lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine,or sodium borate.

In certain embodiments, the vaccine formulation or pharmaceuticalcomposition includes one or more buffers such as a mixture of sodiumbicarbonate and ascorbic acid. In some embodiments, the vaccineformulation may be administered in saline, such as phosphate bufferedsaline (PBS), or distilled water.

In certain embodiments, the vaccine formulation or pharmaceuticalcomposition includes one or more surfactants such as polysorbate 80(Tween 80), Polyethylene glycol tert-octylphenyl ethert-Octylphenoxypolyethoxyethanol4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100);Polyoxyethylenesorbitan monolaurate Polyethylene glycol sorbitanmonolaurate (TWEEN 20); and 4-(1,1,3,3-Tetramethylbutyl)phenol polymerwith formaldehyde and oxirane (TYLOXAPOL). A surfactant can be ionic ornonionic.

In certain embodiments, the vaccine formulation or pharmaceuticalcomposition includes one or more salts such as sodium chloride, ammoniumchloride, calcium chloride, or potassium chloride.

In certain embodiments, a preservative is included in the vaccine. Inother embodiments, no preservative is used. A preservative is most oftenused in multi-dose vaccine vials, and is less often needed insingle-dose vaccine vials. In certain embodiments, the preservative is2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and/orsorbic acid.

In certain embodiments, the vaccine formulation or pharmaceuticalcomposition is a controlled-release formulation.

DNA Vaccines

In certain aspects, the vaccine comprises one or more of the nucleicacids disclosed herein. When a nucleic acid vaccine is administered to apatient, the corresponding gene product (such as a desired antigen) isproduced in the patient's body. In some embodiments, nucleic acidvaccine vectors that include optimized recombinant polynucleotides canbe delivered to a mammal (including humans) to induce a therapeutic orprophylactic immune response. The nucleic acid may be, for example, DNA,RNA, or a synthetic nucleic acid. The nucleic acid may be singlestranded or double-stranded.

Nucleic acid vaccine vectors (e.g., adenoviruses, liposomes,papillomaviruses, retroviruses, etc.) can be administered directly tothe mammal for transduction of cells in vivo. The nucleic acid vaccinescan be formulated as pharmaceutical compositions for administration inany suitable manner, including parenteral administration. Plasmidvectors are typically more efficient for gene transfer to muscle tissue.The potential to deliver DNA vectors to mucosal surfaces by oraladministration has also been reported (PLGA encapsulated Rotavirus andHepatitis B) and DNA plasmids have been utilized for direct introductionof genes into other tissues. DNA vaccines have been introduced intoanimals primarily by intramuscular injection, by gene gun delivery, orby electroporation. After being introduced, the plasmids are generallymaintained episomally without replication. Expression of the encodedproteins has been shown to persist for extended time periods, providingstimulation of B and T cells.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of an infection or other condition, thephysician evaluates vector toxicities, progression of the disease, andthe production of anti-vector antibodies, if any. Often, the doseequivalent of a naked nucleic acid from a vector is from about 1 μg to 1mg for a typical 70 kilogram patient, and doses of vectors used todeliver the nucleic acid are calculated to yield an equivalent amount oftherapeutic nucleic acid. Administration can be accomplished via singleor divided doses. The toxicity and therapeutic efficacy of the nucleicacid vaccine vectors can be determined using standard pharmaceuticalprocedures in cell cultures or experimental animals.

A nucleic acid vaccine can contain DNA, RNA, a modified nucleic acid, ora combination thereof. In some embodiments, the vaccine comprises one ormore cloning or expression vectors; for instance, the vaccine maycomprise a plurality of expression vectors each capable of autonomousexpression of a nucleotide coding region in a mammalian cell to produceat least one immunogenic polypeptide. An expression vector oftenincludes a eukaryotic promoter sequence, such as the nucleotide sequenceof a strong eukaryotic promoter, operably linked to one or more codingregions. The compositions and methods herein may involve the use of anyparticular eukaryotic promoter, and a wide variety are known, such as aCMV or RSV promoter. The promoter can be, but need not be, heterologouswith respect to the host cell. The promoter used may be a constitutivepromoter.

A vector useful in the present compositions and methods can be circularor linear, single-stranded or double stranded and can be a plasmid,cosmid, or episome. In a suitable embodiment, each nucleotide codingregion is on a separate vector; however, it is to be understood that oneor more coding regions can be present on a single vector, and thesecoding regions can be under the control of a single or multiplepromoters.

Numerous plasmids may be used for the production of nucleic acidvaccines. Suitable embodiments of the nucleic acid vaccine employconstructs using the plasmids VR1012 (Vical Inc., San Diego Calif.),pCMVI.UBF3/2 (S. Johnston, University of Texas) or pcDNA3.1 (InVitrogenCorporation, Carlsbad, Calif.) as the vector. In addition, the vectorconstruct can contain immunostimulatory sequences (ISS), such asunmethylated dCpG motifs, that stimulate the animal's immune system. Thenucleic acid vaccine can also encode a fusion product containing theimmunogenic polypeptide. Plasmid DNA can also be delivered usingattenuated bacteria as delivery system, a method that is suitable forDNA vaccines that are administered orally. Bacteria are transformed withan independently replicating plasmid, which becomes released into thehost cell cytoplasm following the death of the attenuated bacterium inthe host cell.

DNA vaccines, including the DNA encoding the desired antigen, can beintroduced into a host cell in any suitable form including, the fragmentalone, a linearized plasmid, a circular plasmid, a plasmid capable ofreplication, an episome, RNA, etc. Preferably, the gene is contained ina plasmid. In certain embodiments, the plasmid is an expression vector.Individual expression vectors capable of expressing the genetic materialcan be produced using standard recombinant techniques. See e.g.,Maniatis et al., 1985 Molecular Cloning: A Laboratory Manual or DNACloning, Vol. I and II (D. N. Glover, ed., 1985) for general cloningmethods.

Routes of administration include, but are not limited to, intramuscular,intranasal, intraperitoneal, intradermal, subcutaneous, intravenous,intraarterially, intraoccularly and oral as well as topically,transdermally, by inhalation or suppository or to mucosal tissue such asby lavage to vaginal, rectal, urethral, buccal and sublingual tissue.Typical routes of administration include intramuscular, intraperitoneal,intradermal and subcutaneous injection. Genetic constructs may beadministered by means including, but not limited to, traditionalsyringes, needleless injection devices, “microprojectile bombardmentgene guns”, or other physical methods such as electroporation (“EP”),“hydrodynamic method”, or ultrasound. DNA vaccines can be delivered byany method that can be used to deliver DNA as long as the DNA isexpressed and the desired antigen is made in the cell.

In some embodiments, a DNA vaccine is delivered via known transfectionreagents such as cationic liposomes, fluorocarbon emulsion, cochleate,tubules, gold particles, biodegradable microspheres, or cationicpolymers. Cochleate delivery vehicles are stable phospholipid calciumprecipitants consisting of phosphatidyl serine, cholesterol, andcalcium; this nontoxic and noninflammatory transfection reagent can bepresent in a digestive system. Biodegradable microspheres comprisepolymers such as poly(lactide-co-glycolide), a polyester that can beused in producing microcapsules of DNA for transfection. Lipid-basedmicrotubes often consist of a lipid of spirally wound two layers packedwith their edges joined to each other. When a tubule is used, thenucleic acid can be arranged in the central hollow part thereof fordelivery and controlled release into the body of an animal.

In some embodiments, DNA vaccine is delivered to mucosal surfaces viamicrospheres. Bioadhesive microspheres can be prepared using differenttechniques and can be tailored to adhere to any mucosal tissue includingthose found in eye, nasal cavity, urinary tract, colon andgastrointestinal tract, offering the possibilities of localized as wellas systemic controlled release of vaccines. Application of bioadhesivemicrospheres to specific mucosal tissues can also be used for localizedvaccine action. In some embodiments, an alternative approach for mucosalvaccine delivery is the direct administration to mucosal surfaces of aplasmid DNA expression vector which encodes the gene for a specificprotein antigen.

The DNA plasmid vaccines according to the present invention areformulated according to the mode of administration to be used. In someembodiments where DNA plasmid vaccines are injectable compositions, theyare sterile, and/or pyrogen free and/or particulate free. In someembodiments, an isotonic formulation is preferably used. Generally,additives for isotonicity can include sodium chloride, dextrose,mannitol, sorbitol and lactose. In some embodiments, isotonic solutionssuch as phosphate buffered saline are preferred. In some embodiments,stabilizers include gelatin and albumin. In some embodiments, avasoconstriction agent is added to the formulation. In some embodiments,a stabilizing agent that allows the formulation to be stable at room orambient temperature for extended periods of time, such as LGS or otherpolycations or polyanions is added to the formulation.

In some embodiments, the DNA vaccine may further comprises apharmacologically acceptable carrier or diluent. Suitable carriers forthe vaccine are well known to those skilled in the art and include butare not limited to proteins, sugars, etc. Such carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous carriers are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and antimicrobials include antioxidants,chelating agents, inert gases and the like. Preferred preservativesinclude formalin, thimerosal, neomycin, polymyxin B and amphotericin B.

An alternative approach to delivering the nucleic acid to an animalinvolves the use of a viral or bacterial vector. Examples of suitableviral vectors include adenovirus, polio virus, pox viruses such asalphaviruses, vaccinia, canary pox, and fowl pox, herpes viruses,including catfish herpes virus, adenovirus-associated vector, andretroviruses. Virus-like vectors include virosomes and virus-likeparticles. Exemplary bacterial vectors include attenuated forms ofSalmonella, Shigella, Edwardsiella ictaluri, Yersinia ruckerii, andListeria monocytogenes. In some embodiments, the nucleic acid is avector, such as a plasmid, that is capable of autologous expression ofthe nucleotide sequence encoding the immunogenic polypeptide.

Use of Vaccines

The vaccines described herein may be used for prophylactic and/ortherapeutic treatment of herpes, including HSV-1 and particularly HSV-2.The subject receiving the vaccination may be a male or a female, and maybe a child or adult. In some embodiments, the subject being treated is ahuman. In other embodiments, the subject is a non-human animal.

Prophylactic Use

In prophylactic embodiments, the HSV-2 vaccine is administered to asubject to induce an immune response that can help protect against theestablishment of HSV-2.

In some embodiments, the vaccine compositions of the invention conferprotective immunity, allowing a vaccinated individual to exhibit delayedonset of symptoms or reduced severity of symptoms (e.g., reduced numberof lesions at the onset of infection), as the result of his/her exposureto the vaccine (e.g., a memory response). In certain embodiments, thereduction in severity of symptoms is at least 25%, 40%, 50%, 60%, 70%,80% or even 90%. Some vaccinated individuals may display no symptomsupon contact with, HSV-2, or even no infection by HSV-2. Protectiveimmunity is typically achieved by one or more of the followingmechanisms: mucosal, humoral, or cellular immunity. Mucosal immunity isprimarily the result of secretory IgA (sIGA) antibodies on mucosalsurfaces of the respiratory, gastrointestinal, and genitourinary tracts.The sIGA antibodies are generated after a series of events mediated byantigen-processing cells, B and T lymphocytes, that result in sIGAproduction by B lymphocytes on mucosa-lined tissues of the body. Humoralimmunity is typically the result of IgG antibodies and IgM antibodies inserum. For example, the IgG titer can be raised by 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or even 100-fold ormore following administration of a vaccine formulation described herein.Cellular immunity can be achieved through cytotoxic T lymphocytes orthrough delayed-type hypersensitivity that involves macrophages and Tlymphocytes, as well as other mechanisms involving T cells without arequirement for antibodies. In particular, cellular immunity may bemediated by T_(H)1 cells or T_(H)17 cells. Activation of T_(H)1 cellscan be measured by secretion of IFN-γ, relative to the level of IFN-γreleased in response to a polypeptide that does not generate animmunologic response. In certain embodiments, the amount of IFN-γreleased is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold,50-fold or even 100-fold greater. The primary result of protectiveimmunity is the destruction of HSV-2 viral particles or inhibition ofHSV-2's ability to replicate. In some embodiments, the protectiveimmunity conferred by presentation of antigen before exposure to HSV-2will reduce the likelihood of seroconversion to an HSV-2-positivestatus.

The duration of protective immunity is preferably as long as possible.In certain embodiments, vaccine formulations produce protective immunitylasting six months, one year, two years, five years, ten years, twentyyears or even a lifetime.

In some embodiments, a combination of specific polypeptides may proveefficacious for inhibiting HSV-2 infection or the onset of symptomsdescribed above. An exemplary vaccine formulation for prophylactic usemay comprise a pharmaceutically-acceptable carrier, a first polypeptideconsisting of SEQ ID NOS: 136, a second polypeptide consisting of SEQ IDNO: 1 or 4, and optionally a third polypeptide consisting of the otherof SEQ ID NOS: 1 and 4, or immunogenic fragments thereof. In someembodiments, the second or third polypeptide consists of polypeptidefragments of SEQ ID NO: 1, such as the polypeptides of SEQ ID NOS: 2,8-16, 138 and 139, or immunogenic fragments thereof. In someembodiments, the vaccine formulation for prophylactic use may comprise afirst polypeptide consisting of SEQ ID NO: 136, a second polypeptideconsisting of SEQ ID NO: 4 or SEQ ID NO: 5, a third polypeptide selectedfrom the group consisting of SEQ ID NOS: 2, 8-16, 138 and 139, andoptionally a fourth polypeptide selected from the group consisting ofSEQ ID NOS: 2, 8-16, 138 and 139, or immunogenic fragments thereof.

In other embodiments, a vaccine formulation for prophylactic usecomprises a pharmaceutically-acceptable carrier and a nucleic acidhaving a nucleotide sequence that encodes at least one of SEQ ID NOS: 1,3, 5, 38, 136 or 138, or an immunogenic fragment thereof. For example,the nucleic acids can have a nucleotide sequence comprising at least oneof SEQ ID NOS: 39, 46, 118, 137 or 140, or a fragment thereof thatencodes an immunogenic polypeptide.

Therapeutic Use

In therapeutic applications, the vaccine comprising a polypeptide ornucleic acid of the invention may be administered to a patient sufferingfrom HSV-2, in an amount sufficient to treat the patient. Treating thepatient, in this case, may refer to delaying or reducing symptoms ofHSV-2 in an infected individual. In some embodiments, treating thepatient refers to reducing the duration of lesions, reducing the numberof lesions, reducing the duration of symptoms per episode, and/orotherwise reducing the intensity of symptoms per episode. In certainembodiments, the vaccine reduces the duration or severity of mildsymptoms; in some embodiments, the vaccine reduces the duration orseverity of serious symptoms. In some embodiments, the vaccine reducesviral shedding and therefore the transmissibility of HSV-2 from thevaccinated patient. In certain embodiments, the reductions describedabove are at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or even 90%. Incertain embodiments, the reductions described above include the completecessation of symptoms, viral shedding and/or future outbreaks (e.g., byblocking the ability of the virus to establish latency in sensoryganglia).

In therapeutic embodiments, the HSV-2 vaccine is administered to anindividual post-infection. The HSV-2 vaccine may be administered shortlyafter infection, e.g. before symptoms manifest, or may be administeredduring or after manifestation of symptoms. In some embodiments, theHSV-2 vaccine may prevent endogenous reactivation of earlier infection.In some embodiments, a post-infection vaccine could be administered topatients in high-risk groups.

The duration of therapeutic effects of a vaccine formulation disclosedherein is preferably as long as possible. In certain embodiments,vaccine formulations produce therapeutic effects lasting one month, twomonths, three months, six months, one year, two years, five years, tenyears, twenty years or even a lifetime.

In some embodiments, a combination of specific polypeptides may proveefficacious for treating a patient suffering from HSV-2 as describedabove. An exemplary vaccine formulation for therapeutic use may comprisea pharmaceutically-acceptable carrier, a first polypeptide consisting ofSEQ ID NOS: 136, a second polypeptide consisting of SEQ ID NO: 1 or 4,and optionally a third polypeptide consisting of the other of SEQ IDNOS: 1 and 4, or immunogenic fragments thereof. In some embodiments, thesecond or third polypeptide consists of polypeptide fragments of SEQ IDNO: 1, such as the polypeptides of SEQ ID NOS: 2, 8-16, 138 and 139, orimmunogenic fragments thereof. In some embodiments, the vaccineformulation for therapeutic use may comprise a first polypeptideconsisting of SEQ ID NO: 136, a second polypeptide consisting of SEQ IDNO: 4 or SEQ ID NO: 5, a third polypeptide selected from the groupconsisting of SEQ ID NOS: 2, 8-16, 138 and 139, and optionally a fourthpolypeptide selected from the group consisting of SEQ ID NOS: 2, 8-16,138 and 139, or immunogenic fragments thereof.

In other embodiments, a vaccine formulation for therapeutic usecomprises a pharmaceutically-acceptable carrier and a nucleic acidhaving a nucleotide sequence that encodes at least one of SEQ ID NOS: 1,3, 5, 38, 136 or 138 or an immunogenic fragment thereof. For example,the nucleic acids can have a nucleotide sequence comprising at least oneof SEQ ID NOS: 39, 46, 118, 137 or 140, or a fragment thereof thatencodes an immunogenic polypeptide.

Assaying Vaccination Efficacy

The efficacy of vaccination with the vaccines disclosed herein may bedetermined in a number of ways.

Vaccine efficacy may be assayed in various model systems. Suitable modelsystems used to study HSV-2 include a guinea pig model and a mousemodel, as described in the examples below. Briefly, the animals arevaccinated and then challenged with HSV-2 or the vaccine is administeredto already-infected animals. The response of the animals to the HSV-2challenge or the vaccine is then compared with control animals, usingone of the measures described above. A similar assay could be used forclinical testing of humans. The treatment and prophylactic effectsdescribed above represent additional ways of determining efficacy of avaccine.

In addition, efficacy may be evaluated by in vitro immunization of naïvehuman peripheral blood mononuclear cells (PBMC), where APCs are exposedto the vaccine and then the APCs are co-cultured with naïve T cells fromthe same donor to evaluate the primary response to immunization in atest tube. An activation of the T-cells by 1.5-fold, 2-fold, 5-fold,10-fold, 20-fold, 50-fold or 100-fold or more relative to activation ofT-cells using APCs not exposed to a vaccine, in certain embodiments, isconsidered an adequate response.

Vaccine efficacy may further be determined by viral neutralizationassays. Briefly, animals are immunized and serum is collected on variousdays post-immunization. Serial dilutions of serum are pre-incubated withvirus during which time antibodies in the serum that are specific forthe virus will bind to it. The virus/serum mixture is then added topermissive cells to determine infectivity by a plaque assay. Ifantibodies in the serum neutralize the virus, there are fewer plaquescompared to the control group.

Uses of Pharmaceutical Compositions

Defense Against HSV Infection

The pharmaceutical compositions of the present disclosure are designedto elicit an immune response against HSV-2. Compositions describedherein may stimulate an innate immune response, an antibody response ora cell-mediated immune response, or a combination of these responses, inthe subject to which it is administered. In some embodiments, thecomposition stimulates immune cells at the peripheral site of infectionor sensory ganglia, such as neutrophils, macrophages, and NK cells. Thecomposition may stimulate infiltration by macrophages; production ofantiviral compounds such as nitric oxide, TNF-α, interferons (IFN), andinterleukin 12 (IL-12) by neutrophils; and/or stimulation of NK cells toproduce IFN-γ. IL-2, IFN-α and IFN-β production may also be triggered bythe polypeptides of the present composition, and are believed to aid incontrolling infection.

In some embodiments, the composition comprises antigens that stimulateproduction of neutralizing antibodies. Neutralizing antibodies maytarget the glycoproteins of the viral envelope, which mediate theinteraction of virions with host cell and are responsible forattachment, binding, and entry of HSV-2 into cells. Accordingly, anexemplary composition comprises one or more glycoproteins describedabove or encoded by nucleic acids described above. Immunogenic antigensand/or epitopes as described herein may be administered separately, inseries, or in combination with one another.

In some embodiments, the composition elicits a cell-mediated response,which may involve CD4⁺ T cells, CD8⁺ T cells and/or production ofantiviral cytokines. The composition may trigger IL-17 secretion byT_(H)17 cells. The composition may trigger IFN-γ secretion, for examplethrough the activation of the innate immune response, and mediate CD8⁺ Tcell clearing of the virus. IFN-γ is also secreted by T_(H)1 cells,T_(C) cells, dendritic cells, and NK cells, and the composition maytrigger IFN-γ secretion by any of these cell types. Such activity ofCD8⁺ T cells may be cytolytic, or, alternately, may be regulated byinhibitor molecules on the surface of the neurons which prevent neuronalkilling. CD4⁺ and/or CD8⁺ T cells may play a role in maintaining latencyof the virus, thus preventing reactivation. In some embodiments, thecomposition boosts a CD4⁺ T cell response and/or a CD8⁺ T cell responsethat prevents reactivation of the virus from its latent state.

In some embodiments, the composition blocks the ability of HSV to evadethe host immune response, or, alternately, boosts immune responsesnormally evaded by HSV. In some embodiments, the composition inhibitsHSV-2 from shifting the immunological balance towards tolerance of HSVantigens. HSV-2 may mediate tolerance through T_(H)2 cells. First, HSV-2may induce suppressor T cells, such as CD4⁺ CD25⁺ T cells and Tr1 cellsthat secrete IL-10, a T_(H)2 cytokine T_(H)2 cytokines downregulatecostimulatory molecules and inhibit the maturation and function ofantigen-presenting dendritic cells. In addition, infection with HSV-2inhibits the maturation and migration of dendritic cells, which areessential for efficient induction of CD8⁺ killer T cells. Notably,T_(H)2 cytokines are produced during recurrence of HSV-2 infection, incontrast to T_(H)1 cytokines, which are produced during recurrence-freeepisodes. Thus, in certain embodiments, the compositions of theinvention repress suppressor T cells and/or induce maturation ormigration or both of dendritic cells.

In some embodiments, methods of inducing an immune response againstHSV-2 in a mammal comprise administering the compositions describedabove. The composition may be used to induce an immune response atdifferent time points, such as before exposure to HSV-2, after initialinfection with HSV-2, before or after HSV-2 has established latency,before or after HSV-2 shedding occurs, and/or before or after recurrentoutbreaks occur. In some embodiments, an immune response against HSV-2may be induced at one or more of the timepoints above. The compositionmay induce a T_(H)1 response and/or a T_(H)17 response but not a T_(H)2response, or may activate the responses at the same time or at differenttimes.

In some embodiments, administration of the composition reduces symptomsassociated with initial infection, latency, or recurrent infection withHSV. Such a composition may reduce incidence and/or severity of lesions,sores, pain, irritation, itching, fever, malaise, headache, viralshedding, or prodromes associated with HSV infection or outbreak.

In some embodiments, one or more antibodies to antigens of HSV-2 may beadministered to individuals in order to produce passive immunity.Passive immunity results from the transfer of active humoral immunity inthe form of ready-made antibodies, from one individual to another.Passive immunization may be used when there is a high risk of infectionand insufficient time for the body to develop its own immune response,or to reduce the symptoms of ongoing or immunosuppressive diseases.Adoptive transfer of T cells may provide another method of eliciting animmune response to HSV-2 antigens in patients. In one embodiment,autologous T cells may be expanded on APCs presenting the antigensderived from the polypeptides described above. Subsequently, theexpanded HSV-2-specific T cells are transferred back into the patientfrom which the T cells were derived.

Diagnostic Uses

This application provides, inter alia, a rapid, inexpensive, sensitive,and specific method for detection of HSV-2 in patients. In this respectit should be useful to hospitals and physicians examining and treatingpatients with or at risk for HSV-2 infection. As used herein, “patient”refers to an individual (such as a human) that either has an HSV-2infection or has the potential to contract an HSV-2 infection.

In some embodiments, one may use an antibody against one of thepolypeptides described herein, such as those of Table 1 and/or Table 2,to detect HSV-2 in an individual. The instant disclosure also provides amethod of phenotyping biological samples from patients suspected ofhaving a HSV-2 infection that involves: (a) rendering a biologicalsample amenable to immunoassay, if necessary; (b) contacting the samplewith an appropriate HSV-2-specific antibody or antigen-binding portionthereof under conditions that allow for binding of the antibody orantigen-binding portion to an epitope of HSV-2; and (c) determining ifthe sample shows the presence of HSV-2 as compared to a control tissue;where if the test tissue shows the presence of HSV-2, the patient isidentified as likely having a HSV-2 infection.

Alternatively, one may use the polypeptides described above to detectanti-HSV-2 antibodies in an individual. The instant disclosure alsoprovides a method of phenotyping biological samples from patientssuspected of having a HSV-2 infection: (a) rendering a biological sampleamenable to an affinity assay such as ELISA, if necessary; (b)contacting the sample with a HSV-2-specific antigen or portion thereofunder conditions that allow for binding of the antigen to any hostantibodies present in the sample; and (c) determining if the sampleshows the presence of HSV-2 as compared to a control tissue; where ifthe test tissue shows the presence of HSV-2, the patient is identifiedas likely having a HSV-2 infection. The aforementioned test may beappropriately adjusted to detect other viral infections, for instance byusing a homolog (from another viral species) of the proteins describedabove, such as in Table 1 and/or Table 2.

A number of methods for measuring antibody-antigen binding are known inthe art, including ELISA (enzyme-linked immunosorbent assay), Westernblotting, competition assay, and spot-blot. The detection step may be,for instance, chemiluminescent, fluorescent, or colorimetric. Onesuitable method for measuring antibody-protein binding is the LuminexxMAP system, where peptides are conjugated to a dye-containingmicrosphere. Certain systems, including the xMAP system, are amenable tomeasuring several different markers in multiplex, and could be used tomeasure levels of antibodies at once. In some embodiments, other systemsare used to assay a plurality of markers in multiplex. For example,profiling may be performed using any of the following systems: antigenmicroarrays, bead microarrays, nanobarcodes particle technology, arrayedproteins from cDNA expression libraries, protein in situ array, proteinarrays of living transformants, universal protein array, lab-on-a-chipmicrofluidics, and peptides on pins. Another type of clinical assay is achemiluminescent assay to detect antibody binding. In some such assays,including the VITROS Eci anti-HCV assay, antibodies are bound to asolid-phase support made up of microparticles in liquid suspension, anda surface fluorometer is used to quantify the enzymatic generation of afluorescent product.

In other embodiments, one may use the polypeptides described above, suchas those of Table 1 and/or Table 2, to detect T cells that are specificto HSV-2. The instant disclosure provides a method of phentoypingbiological samples from patients suspected of having a HSV-2 infection,involving (a) rendering a biological sample amenable to an assay foractivation of T cells, if necessary, (b) contacting the sample with aHSV-2-specific polypeptide or portion thereof under conditions thatallow APCs to process the polypeptide, and (c) determining activation ofthe T cells in response to the HSV-2-specific polypeptide, where anelevated T cell activation relative to an uninfected patient indicatesHSV-2 infection. This diagnostic assay is intended to detect thepresence of HSV-2-specific T cells in any patients, including thosepatients who have been exposed to HSV-2 but have not seroconverted toproduce detectable levels of anti-HSV-2 antibodies.

T cell activation may be measured using many assays, includingcytokine-specific ELISA, cell proliferation measured by tritiatedthymidine incorporation or membrane intercolating (PKH-67) orcytoplasmic (CFSE) dyes, ELISPOT, flow cytometry, and bead arrays. Inaddition, one may measure the T cell response in T cell lines or in Tcell hybridomas from mice or humans that are specific for the antigens.Readouts for activated T cells include proliferation, cytokineproduction, or readout of a surrogate enzyme expressed by the hybridomathat is induced when the T cell or T cell hybridoma is activated inresponse to an antigen. For example, activation of a T cell response maybe detected by T cell hybridoma that is engineered to produceβ-galactosidase. β-galactosidase may be detected through the use ofcolorimetric β-galactosidase substrates such as chlorophenyl red β-Dgalactopyranoside (CPRG).

Infection with HSV-2 may be acute or latent. In some embodiments, if thebiological sample shows the presence of HSV-2, one may administer atherapeutically effective amount of the compositions and therapiesdescribed herein to the patient. The biological sample may comprise, forexample, blood, semen, urine, vaginal fluid, mucus, saliva, feces,urine, cerebrospinal fluid, or a tissue sample. In some embodiments, thebiological sample is an organ intended for transplantation. In certainembodiments, before the detection step, the biological sample is subjectto culture conditions that promote the growth of HSV-2.

The diagnostic tests herein may be used to detect HSV-2 in a variety ofsamples, including samples taken from patients and samples obtained fromother sources. For example, the diagnostic tests may be used to detectHSV-2 on objects such as medical instruments. In some embodiments, thetests herein may be performed on samples taken from animals such asagricultural animals (cows, pigs, chickens, goats, horses and the like),companion animals (dogs, cats, birds, and the like), or wild animals. Incertain embodiments, the tests herein may be performed on samples takenfrom cell cultures such as cultures of human cells that produce atherapeutic protein, cultures of bacteria intended to produce a usefulbiological molecule, or cultures of cells grown for research purposes.

The invention also includes a method of determining the location of aHSV-2 infection in a patient comprising: (a) administering apharmaceutical composition comprising a labeled HSV-2 antibody orantigen-binding portion thereof to the patient, (b) detecting the label,and (c) determining if the patient has HSV-2 compared to a control. Incertain embodiments, the method further comprises, if the patient has anHSV-2 infection, administering a therapeutically effective amount of acomposition described herein to the patient. The method may furthercomprise determining the infected cell types and/or volume of the HSV-2in the patient. This method may be used to evaluate the spread of HSV-2in the patient and determine whether a localized therapy is appropriate.

In some embodiments, the polypeptides described herein may be used tomake a prognosis of the course of infection. In some embodiments, T cellor antibody responses specific for the polypeptides herein may bedetected in a sample taken from a patient. If antibodies or T cells arepresent at normal levels, it would indicate that the patient has raisedan effective immune response against the pathogen. If antibodies or Tcells are absent, or present at reduced levels, it would indicate thatthe patient is failing to raise a sufficient response against thepathogen, and a more aggressive treatment would be recommended. In someembodiments, antibody or T cells present at reduced levels refers toresponses that are present at less than 50%, 20%, 10%, 5%, 2%, or 1% thetypical level in a patient with a protective immune response. T cellresponses may be detected by methods known in the art such as T cellproliferation, ELISPOT or ELISA, and antibodies may be detected byaffinity for any of the antigens described herein, using methods knownin the art such as ELISA.

In some embodiments, detection of T cells specific for HSV-2 antigensmay be used to predict the progress and symptoms of HSV-2 infection in apatient. After infection with HSV-2, some patients remain asymptomatic,although the virus may establish latency. Other patients exhibitsymptoms of HSV-2 infection, and may experience recurrent outbreaks. TheHSV-2 antigens found in asymptomatic patients may differ from thoseantigens found in patients who present symptoms and/or recurrentoutbreaks. Accordingly, the detection methods of the present inventionmay be used to distinguish between subgroups within the population ofpatients infected with HSV-2. Subgroups may be further divided intopatients who experience frequent outbreaks and those who infrequently ornever experience outbreaks, or patients who shed high levels of virusand those who shed low levels or do not shed. The categorization ofpatients, based on the presence and levels of T cell responses tocertain HSV-2 antigens but not others, may help health carepractitioners to determine appropriate treatment regimens. Similarly,differences in the magnitude of T cell responses and/or differences inthe combination and levels of cytokines produced by T cells may also beused to predict the progress and symptoms of HSV-2 infection in apatient. Thus, an infected patient whose complement of HSV-2 antigens towhich T cells respond predicts severe symptoms, frequent outbreaks,and/or high levels of viral shedding may require more intensiveantiviral therapy and/or a longer course of therapeutic treatment than apatient whose complement of HSV-2 antigens predicts an asymptomaticinfection.

It will be understood by one of skill in the art that the methods hereinare not limited to detection of HSV-2. Other embodiments include thedetection of related viruses including viruses with proteins homologousto the proteins described above, such as those in Table 1 and/or Table2. Such related viruses include, for example, other members of theHerpesviridae family. Depending on the homology, these related virusesmay also include viruses that are not members of the Herpesviridaefamily.

Use in Groups with Increased Risk for Infection by HSV-2

Essentially any individual has a certain risk of infection with HSV-2.However, certain sub-populations have an increased risk of infection. Insome embodiments, patients receiving the composition for HSV-2 areimmunocompromised.

An immunocompromising condition arising from a medical treatment islikely to expose the individual in question to a higher risk ofinfection. It is possible to treat an infection prophylactically in anindividual having the immunocompromised condition before or duringtreatments known to generate such a condition. By prophylacticallytreating with the antigen before or during a treatment known to generatesuch a condition it is possible to prevent a subsequent infection or toreduce the risk of the individual contracting an infection due to theimmunocompromised condition. Should the individual contract aninfection, e.g., following a treatment leading to an immunocompromisedcondition, it is also possible to treat the infection by administeringto the individual an antigen composition.

In certain embodiments, the compositions are administered to children oradult patients. In other embodiments, compositions are appropriate forpregnant women who were infected before becoming pregnant, or who becameinfected during pregnancy, such as to inhibit infection of a fetus orbaby. The compositions may also be administered to neonates and infantswho became infected in utero or during delivery.

Doses and Routes of Administration

Dosage Amounts and Timing

The amount of antigen in each vaccine dose is selected as an effectiveamount, which induces a prophylactic or therapeutic response, asdescribed above, in either a single dose or over multiple doses.Preferably, the dose is without significant adverse side effects intypical vaccines. Such amount will vary depending upon which specificantigen is employed. Generally, it is expected that a dose will comprise1-1000 μg of protein, in some instances 2-100 μg, for instance 4-40 μg.Alternatively, a dose will comprise 10-6000 μg of nucleic acid, in someinstances 20-4000 μg, for instance 30-4000 μg. An optimal amount for aparticular vaccine can be ascertained by standard studies involvingobservation of antibody titers, T cell activation levels, and otherresponses in subjects. In some embodiments, the appropriate amount ofantigen to be delivered will depend on the age, weight, and health(e.g., immunocompromised status) of a subject. When present, typicallyan adjuvant will be present in amounts from 1 μg-250 μg per dose, forexample 50-150 μg, 75-125 μg or 100 μg.

In some embodiments, only one dose of the vaccine is administered toachieve the results described above. In other embodiments, following aninitial vaccination, subjects receive one or more boost vaccinations,for a total of two, three, four or five vaccinations. Advantageously,the number is three or fewer. A boost vaccination may be administered,for example, about 1 month, 2 months, 4 months, 6 months, or 12 monthsafter the initial vaccination, such that one vaccination regimeninvolves administration at 0, 0.5-2 and 4-8 months. It may beadvantageous to administer split doses of vaccines which may beadministered by the same or different routes.

In some embodiments, the invention supplies a treatment regimencomprising a first dose of vaccine and a second, third or fourth dose ofvaccine (a boost vaccine). In exemplary embodiments, a first dose ofvaccine comprises one or more polypeptide antigens, or nucleic acidsencoding one or more polypeptide antigens, or a combination of one ormore polypeptide antigens and nucleic acids encoding the same or otherprotein antigens. In some embodiments, a boost vaccine is formulatedwith the same polypeptide antigens, nucleic acids, or polypeptideantigens and nucleic acids as the first dose. In some embodiments, aboost vaccine is formulated with different polypeptide antigens, nucleicacids, or polypeptide antigens and nucleic acids from the first dose. Insome embodiments, the first dose may comprise only polypeptide antigensand boost vaccine may comprise only nucleic acids, or the first dose maycomprise only nucleic acids and boost vaccine may comprise onlypolypeptide. In some embodiments, the first dose may comprisepolypeptide antigens and nucleic acids, and boost vaccine may compriseonly protein antigens or only nucleic acids. In some embodiments, thefirst dose may comprise only protein antigens or only nucleic acids, andboost vaccine may comprise protein antigens and nucleic acids. Incertain embodiments where the boost vaccine is a polypeptide, thepolypeptide is gL2 (SEQ ID NO: 3) or ICP4 (SEQ ID NO: 1) or animmunogenic fragment thereof (e.g., ICP4.2, and gL2s v.2, SEQ ID NOS: 2and 136), optionally in combination with one or more of the adjuvantsdescribed above, particularly one or more of the ISCOMs. Suchpolypeptide boost vaccines are particularly useful in conjunction withany one of the nucleic acid vaccines described above (e.g., nucleicacids having nucleotide sequences that encode at least one of SEQ IDNOS: 1, 3, 5, 38, 136 or 138, or an immunogenic fragment thereof).

The pharmaceutical compositions described herein may take on a varietyof dosage forms. In certain embodiments, the composition is provided insolid or powdered (e.g., lyophilized) form; it also may be provided insolution form. In certain embodiments, a dosage form is provided as adose of lyophilized composition and at least one separate sterilecontainer of diluent.

In some embodiments, the antigen is delivered to a patient at an amountof 1 μmol per dose. In some embodiments, the antigen is delivered at adose ranging from 10 nmol to 100 nmol per dose. The appropriate amountof antigen to be delivered may be determined by one of skill in the art.In some embodiments, the appropriate amount of antigen to be deliveredwill depend on the age, weight, and health (e.g., immunocompromisedstatus) of a subject.

Pharmaceutical compositions disclosed herein are (in some embodiments)administered in amounts sufficient to elicit production of antibodies aspart of an immunogenic response. In some embodiments, the compositionmay be formulated to contain 5 μg/0.5 ml or an amount ranging from 10μg/1 ml to 200 μg/1 ml of an antigen. In other embodiments, thecomposition may comprise a combination of antigens. The plurality ofantigens may each be the same concentration, or may be differentconcentrations.

In some embodiments, the composition will be administered in a doseescalation manner, such that successive administrations of thecomposition contain a higher concentration of composition than previousadministrations. In some embodiments, the composition will beadministered in a manner such that successive administrations of thecomposition contain a lower concentration of composition than previousadministrations.

In therapeutic applications, compositions are administered to a patientsuffering from a disease in an amount sufficient to cure or at leastpartially arrest the disease and its complications.

Therapeutic applications of a composition described herein includereducing transmissibility, slowing disease progression, reducing viralshedding, or eliminating recurrent infections in patients that have beeninfected with HSV-2, such as by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%or 10% of the levels at which they would occur in individuals who arenot treated with the composition. The composition may also reduce thequantity of HSV-2 shed by infected individuals, inhibit the expressionof proteins required for reactivation of HSV-2 from the latent stage ininfected patients, and/or inhibit replication of HSV-2 in neurons ofinfected patients, such as by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or10% of the levels at which they would occur in individuals not treatedwith the composition.

In prophylactic embodiments, compositions are administered to a human orother mammal to induce an immune response that can inhibit theestablishment of an infectious disease or other condition. In someembodiments, a composition may partially block the virus fromestablishing latency or reduce the efficiency with which latency isestablished.

In some embodiments, only one dose (administration) of the compositionis given. In other embodiments, the composition is administered inmultiple doses. In various embodiments, the composition is administeredonce, twice, three times, or more than three times. The number of dosesadministered to a subject is dependent upon the antigen, the extent ofthe disease or the expected exposure to the disease, and the response ofa subject to the composition.

In some embodiments, the compositions are administered in combinationwith antimicrobial molecules. Antimicrobial molecules may includeantiviral molecules. Many antiviral molecules are currently known in theart, and target one or more stage of the viral life cycle, includingviral attachment to host cells, release of viral genes and/or enzymesinto the host cell, replication of viral components using host-cellmachinery, assembly of viral components into complete viral particles,and release of viral particles to infect new hosts.

Routes of Administration

The vaccine formulations and pharmaceutical compositions herein can bedelivered by administration to an individual, typically by systemicadministration (e.g., intravenous, intraperitoneal, intramuscular,intradermal, subcutaneous, transdermal, subdermal, intracranial,intranasal, mucosal, anal, vaginal, oral, sublingual, buccal route orthey can be inhaled) or they can be administered by topical application.

In some embodiments, the composition may be administered directly to thelikely sites of infection. In female patients, the composition may beapplied topically to mucosal membranes, or delivered vaginally orrectally using devices and methods known in the art. The vaginal andrectal routes of delivery permit extended, continuous or pulsed deliveryand administration of composition dosages, and may be administeredeither before or after exposure to HSV, depending on the use of aprophylactic or therapeutic composition. In male patients, thecomposition may be applied topically to the skin or mucosal membranes,or delivered rectally. In both patient populations, the composition mayalso be targeted to the sensory ganglia.

An HSV-2 vaccine or pharmaceutical composition is often administered viathe intramuscular route. Typically, in this route, the vaccine isinjected into an accessible area of muscle tissue. Intramuscularinjections are, in some embodiments, given in the deltoid, vastuslateralis, ventrogluteal or dorsogluteal muscles. The injection istypically given at an approximately 90° angle to the surface of theskin, so the vaccine penetrates the muscle.

An HSV-2 vaccine may also be administered subcutaneously. The injectionis typically given at a 45° angle to the surface of the skin, so thevaccine is administered to the subcutis and not the muscle.

In some embodiments, the HSV-2 vaccine is administered intradermally.Intradermal administration is similar to subcutaneous administration,but the injection is not as deep and the target skin layer is thedermis. The injection is typically given at a 10-15° angle to thesurface of the skin, so the vaccine is delivered just beneath theepidermis.

In some embodiments, the HSV-2 vaccine is administered byelectroporation. Delivery by electroporation may be intramuscular orintradermal. Suitable devices for electroporation include devices madeby Inovio Pharmaceuticals, Inc. (Blue Bell, Pa.) and the TriGrid™Delivery System made by Ichor Medical Systems, Inc. (San Diego, Calif.).

Formulations

The vaccine formulation may be suitable for administration to a humanpatient, and vaccine preparation may conform to USFDA guidelines. Insome embodiments, the vaccine formulation is suitable for administrationto a non-human animal. In some embodiments, the vaccine is substantiallyfree of either endotoxins or exotoxins. Endotoxins include pyrogens,such as lipopolysaccharide (LPS) molecules. The vaccine may also besubstantially free of inactive protein fragments. In some embodiments,the vaccine has lower levels of pyrogens than industrial water, tapwater, or distilled water. Other vaccine components may be purifiedusing methods known in the art, such as ion-exchange chromatography,ultrafiltration, or distillation. In other embodiments, the pyrogens maybe inactivated or destroyed prior to administration to a patient. Rawmaterials for vaccines, such as water, buffers, salts and otherchemicals may also be screened and depyrogenated. All materials in thevaccine may be sterile, and each lot of the vaccine may be tested forsterility. Thus, in certain embodiments the endotoxin levels in thevaccine fall below the levels set by the USFDA, for example 0.2endotoxin (EU)/kg of product for an intrathecal injectable composition;5 EU/kg of product for a non-intrathecal injectable composition, and0.25-0.5 EU/ml for sterile water.

In some embodiments, the vaccine comprising a polypeptide contains lessthan 5%, 2%, 1%, 0.5%, 0.2%, 0.1% of other, undesired unpolypeptides,relative to the amount of desired polypeptides. In some embodiments, thevaccine contains less than 5%, less than 2%, less than 1%, less than0.5%, less than 0.2%, or less than 0.1% DNA and/or RNA.

It is preferred that the vaccine has low or no toxicity, within areasonable risk-benefit ratio.

The formulations suitable for introduction of the pharmaceuticalcomposition vary according to route of administration. Formulationssuitable for parenteral administration, such as, for example, byintraarticular (in the joints), intravenous, intramuscular, intradermal,intraperitoneal, intranasal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by the packaged nucleic acid can also be administeredintravenously or parenterally.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the polypeptides or packagednucleic acids suspended in diluents, such as water, saline or PEG 400;(b) capsules, sachets or tablets, each containing a predetermined amountof the active ingredient, as liquids, solids, granules or gelatin; (c)suspensions in an appropriate liquid; and (d) suitable emulsions. Tabletforms can include one or more of lactose, sucrose, mannitol, sorbitol,calcium phosphates, corn starch, potato starch, tragacanth,microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, fillers, binders, diluents, buffering agents,moistening agents, preservatives, flavoring agents, dyes, disintegratingagents, and pharmaceutically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art. The pharmaceutical compositionscan be encapsulated, e.g., in liposomes, or in a formulation thatprovides for slow release of the active ingredient.

The antigens, alone or in combination with other suitable components,can be made into aerosol formulations (e.g., they can be “nebulized”) tobe administered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

Suitable formulations for vaginal or rectal administration include, forexample, suppositories, which consist of the polypeptides or packagednucleic acids with a suppository base. Suitable suppository basesinclude natural or synthetic triglycerides or paraffin hydrocarbons. Inaddition, it is also possible to use gelatin rectal capsules whichconsist of a combination of the polypeptides or packaged nucleic acidswith a base, including, for example, liquid triglycerides, polyethyleneglycols, and paraffin hydrocarbons. The formulation may be suitable foradministration to a human patient, and the preparation may conform to USFDA guidelines. In some embodiments, the formulation is suitable foradministration to a non-human animal. In some embodiments, thecomposition is substantially free of either endotoxins or exotoxins.Endotoxins may include pyrogens, such as lipopolysaccharide (LPS)molecules. The composition may also be substantially free of inactiveprotein fragments which may cause a fever or other side effects. In someembodiments, the composition contains less than 1%, less than 0.1%, lessthan 0.01%, less than 0.001%, or less than 0.0001% of endotoxins,exotoxins, and/or inactive protein fragments. In some embodiments, thecomposition has lower levels of pyrogens than industrial water, tapwater, or distilled water. Other components may be purified usingmethods known in the art, such as ion-exchange chromatography,ultrafiltration, or distillation. In other embodiments, the pyrogens maybe inactivated or destroyed prior to administration to a patient. Rawmaterials for compositions, such as water, buffers, salts and otherchemicals may also be screened and depyrogenated. All materials in thecomposition may be sterile, and each lot of the composition may betested for sterility. Thus, in certain embodiments the endotoxin levelsin the composition fall below the levels set by the USFDA: 0.2 endotoxin(EU)/kg of product for an intrathecal injectable composition; 5 EU/kg ofproduct for a non-intrathecal injectable composition, and 0.25-0.5 EU/mlfor sterile water.

In certain embodiments, the preparation comprises less than 50%, 20%,10%, or 5% (by dry weight) contaminating protein. In certainembodiments, the desired molecule is present in the substantial absenceof other biological macromolecules, such as other proteins (particularlyother proteins which may substantially mask, diminish, confuse or alterthe characteristics of the component proteins, either as purifiedpreparations or in their function in the subject reconstituted mixture).In certain embodiments, at least 80%, 90%, 95%, 99%, or 99.8% (by dryweight) of biological macromolecules of the same type present (butwater, buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present).

It is preferred that the composition has low or no toxicity, within areasonable risk-benefit ratio. In certain embodiments, the compositioncomprises ingredients at concentrations that are less than LD₅₀measurements for the animal being treated with the composition. LD₅₀measurements may be obtained in mice or other experimental modelsystems, and extrapolated to humans and other animals. Methods forestimating the LD₅₀ of compounds in humans and other animals arewell-known in the art. A composition, and any component within it, mighthave an LD₅₀ value in rats of greater than 100 g/kg, greater than 50g/kg, greater than 20 g/kg, greater than 10 g/kg, greater than 5 g/kg,greater than 2 g/kg, greater than 1 g/kg, greater than 500 mg/kg,greater than 200 mg/kg, greater than 100 mg/kg, greater than 50 mg/kg,greater than 20 mg/kg, or greater than 10 mg/kg. In some embodiments,the therapeutic index of the composition (measured as the toxic dose for50% of the population (TD₅₀) divided by the minimum effective dose for50% of the population (ED₅₀)), is greater than 1, greater than 10, orgreater than 100.

Preparation and Storage of Vaccines Formulations and ImmunogenicCompositions

The HSV-2 vaccines described herein may be produced using a variety oftechniques. For example, a polypeptide may be produced using recombinantDNA technology in a suitable host cell. A suitable host cell may bebacterial, yeast, mammalian, or other type of cell. The host cell may bemodified to express an exogenous copy of one of the relevant polypeptidegenes. Typically, the gene is operably linked to appropriate regulatorysequences such as a strong promoter and a polyadenylation sequence. Insome embodiments, the promoter is inducible or repressible. Otherregulatory sequences may provide for secretion or excretion of thepolypeptide of interest or retention of the polypeptide of interest inthe cytoplasm or in the membrane, depending on how one wishes to purifythe polypeptide. The gene may be present on an extrachromosomal plasmid,or may be integrated into the host genome. One of skill in the art willrecognize that it is not necessary to use a nucleic acid 100% identicalto the naturally-occurring sequence. Rather, some alterations to thesesequences are tolerated and may be desirable. For instance, the nucleicacid may be altered to take advantage of the degeneracy of the geneticcode such that the encoded polypeptide remains the same. In someembodiments, the gene is codon-optimized to improve expression in aparticular host. The nucleic acid may be produced, for example, by PCRor by chemical synthesis.

Once a recombinant cell line has been produced, a polypeptide may beisolated from it. The isolation may be accomplished, for example, byaffinity purification techniques or by physical separation techniques(e.g., a size column).

In a further aspect of the present disclosure, there is provided amethod of manufacture comprising mixing one or more polypeptides or animmunogenic fragment or variant thereof with a carrier and/or anadjuvant. In some embodiments, the adjuvant is one that stimulates aT_(H)1 cell response.

In some embodiments, antigens for inclusion in compositions of theinvention may be produced in cell culture. One method comprisesproviding one or more mammalian expression vectors and cloningnucleotides encoding two or more polypeptides selected from polypeptideshaving an amino acid sequence of any one of SEQ ID NOS: 1-38, 135, 136,138 or 139, then expressing and isolating the polypeptides.

In some embodiments, nucleic acids for inclusion in compositions of theinvention may be produced by replication in a bacterial host such as E.coli and purified by standard RNA or DNA purification methods.

The immunogenic polypeptides described herein, and nucleic acidcompositions that express the polypeptides, can be packaged in packs,dispenser devices, and kits for administering nucleic acid compositionsto a mammal. For example, packs or dispenser devices that contain one ormore unit dosage forms are provided. Typically, instructions foradministration of the compounds will be provided with the packaging,along with a suitable indication on the label that the compound issuitable for treatment of an indicated condition, such as thosedisclosed herein.

EXEMPLIFICATION Example 1 Identification of HSV-2 Antigens

A library of HSV-2 polypeptides (from HSV-2 Strain G, Lot #7C0013, fromAdvanced Biotechnologies Inc, Maryland) was prepared and screened withperipheral blood mononuclear cells (PBMC) from human donors. Briefly, alibrary of HSV polypeptides was expressed by bacteria and mixed withAPCs. The APCs, in turn, presented HSV-derived peptides to lymphocytesthat had been isolated from human patients infected with HSV-2. Thepatients belonged to several populations, as described below. Lymphocyteresponses from each population were compared for reactivity to eachexpressed protein, and the screen detected antigens that inducedreactive lymphocytes with greater frequency in one patient population ascompared to the others. Infected but asymptomatic, and exposed butseronegative patients may activate protective immune responses thatpatients who experience frequent outbreaks do not; in particular,exposed but seronegative patients are presumed to have mountedsterilizing immunity to HSV-2 infection. It is believed that a uniqueset of polypeptides will activate lymphocytes from these patientpopulations.

The release of IFN-γ from CD4⁺ T cells and CD8⁺ T cells from eachpopulation was measured by ELISA following exposure to candidateantigens. Antigens were selected on the basis of the fold increase ofIFN-γ released, relative to the level of IFN-γ released by frequentrecurrers who experience more than four outbreaks per year, as well asthe frequency of responders in the infected but asymptomatic, or exposedbut seronegative populations, compared to frequent and less-frequentrecurrers.

Identification of Antigens Encoded by UL10, UL19, UL40, US4, US6, RS1(RS1.1, RS1.2, RS1.3), UL 36 (UL36.3, UL36.4, UL36.5), UL32, and RL2

Lymphocytes were isolated from patients belonging to severalpopulations: infected but asymptomatic (n=40), exposed but seronegative(n=40), frequent recurrers who experience four or more outbreaks peryear (n=43), less-frequent recurrers who experience less than fouroutbreaks per year (n=19), naïve (n=10), and HSV-2⁻/HSV-1⁺ (n=10). Table3 shows the frequency analysis for thirteen HSV-2 antigens encoded byUL10, UL19, UL40, US4, US6, RS1 (RS1.1, RS1.2, RS1.3), UL36 (UL36.3, UL36.4, UL36.5), UL32, and RL2 in the exposed patient cohort compared tothe recurrer cohorts (frequent and less-frequent recurrers combined).

TABLE 3 Frequency analysis for antigens encoded by UL10, UL19, UL40,US4, US6, RS1 (RS1.1, RS1.2, RS1.3), UL36 (UL36.3, UL36.4, UL36.5), UL32and RL2 Frequency Analysis (HSV-1/HSV-2 seronegative) % response fromfold increase over HSV-2 Gene Protein Name exposed donors recurrerresponse UL10 gM2 23% 1.4 UL19 VP5 — — UL40 ribonucleotide 36% 3.0reductase US4 gG2 24% 1.6 US6 gD2 27% 1.9 RS1 ICP4 RS1.1 54% 3.0 RS1.246% 2.3 RS1.3 23% 1.2 UL36 Major tegument UL36.3 protein 46% 2.3 UL36.446% 4.2 UL36.5 31% 1.9 UL32 DNA cleavage & — — packaging proteiin RL2ICP0 45% 1.6Identification of Antigens Encoded by UL1, UL49.5, and UL54

Lymphocytes were isolated from patients belonging to severalpopulations: infected but asymptomatic (n=40), exposed but seronegative(n=40), frequent recurrers who experience four or more outbreaks peryear (n=43), less-frequent recurrers who experience less than fouroutbreaks per year (n=19), naïve (n=10), and HSV-2⁻/HSV-1⁺ (n=10).

Table 4 shows the frequency analysis for three HSV-2 antigens encoded byUL1, UL49.5 and UL54, in the exposed patient cohort compared to therecurrer cohorts (frequent and less-frequent recurrers combined).

TABLE 4 Frequency analysis for antigens encoded by UL1, UL49.5, and UL54Frequency Analysis (HSV-1/HSV-2 seronegative) Protein % response fromfold increase over HSV-2 Gene Name exposed donors recurrer response UL1gL2s v.2 64% 2.7 UL49.5 (virion p) 37% 2.1 UL54 ICP27 22% 5.8Identification of Antigens Encoded by RL1, UL2, and UM

Lymphocytes were isolated from patients belonging to severalpopulations: infected but asymptomatic (n=40), exposed but seronegative(n=40), frequent recurrers who experience four or more outbreaks peryear (n=43), less-frequent recurrers who experience less than fouroutbreaks per year (n=19), naïve (n=10), and HSV-2⁻/HSV-1⁺ (n=10).

Table 5 shows the frequency analysis for three HSV-2 antigens encoded byRL1, UL2, and UL11 in the exposed patient cohort compared to therecurrer cohorts (frequent and less-frequent recurrers combined).

TABLE 5 Frequency analysis for HSV-2 antigens encoded by RL1, UL2, andUL11 Frequency Analysis (HSV-1/HSV-2 seronegative) Protein % responsefrom fold increase over HSV-2 Gene Name exposed donors recurrer responseRL1 ICP34.5 45% 1.3 UL2 DNA 23% 1.4 glycosylase UL11 tegument 21% <1.0protein

Example 2 In Vivo Data: Protein Antigen Immunizations

Guinea Pig Therapeutic Vaccination Protocol [Protocol A]

Female Hartley guinea pigs were challenged intravaginally with HSV-2strain MS at 5×10⁵ pfu to establish a genital tract infection. Animalswere monitored for infection by vaginal swab on day 1 post-infection,and acute disease between days 3 and 14 post-infection. On day 14, afterresolution of primary disease, the animals were randomized into groupsof 12 and immunized subcutaneously with antigen (each HSV-2 polypeptideat 15 μg dose) plus adjuvant (50 μg dose of an ISCOM matrix with a 91:9mixture of Quillaja saponin matrices A and C). Each group received atotal of 3 vaccinations, on days 14, 21, and 35 post-infection. Genitalswabs were collected during the vaccination period to monitor viralshedding, and daily observations were recorded. Symptoms were scored ona scale from 0 to 4 based upon severity, 0=no symptoms; 1=redness orswelling; 2=a few small vesicles; 3=several large vesicles; 4=severallarge vesicles with maceration. In addition, animals with lesionsintermediate in severity between the above scores were given a score of0.5, 1.5, 2.5, or 3.5.

Exemplary Therapeutic Vaccination Studies with ICP4.2, gD2ΔTMR, and gD2

The results of exemplary studies are presented below in Tables 6-10. Theneutralizing antibody titer was determined at day 41 post-infection and7 days after third immunization using an average of 4 out of the 12-20animals in each group. The mean recurrent lesion scores and mean lesiondays were each determined from day 15 to day 63 or day 70post-infection. The lesion scores represent total lesions for each groupfrom day 15 to 60 and then a mean was calculated. Mean lesion daysrepresent the mean number of days post-infection that immunized ornon-immunized animals had herpetic lesions present. Vaginal-swab sampleswere collected from all animals for 12 days between days 20-63post-infection and stored at −80° C. until assayed for virus sheddingtiters by quantitative real-time PCR.

TABLE 6 Results of therapeutic vaccination studies with ICP4.2 (SEQ IDNO: 2): lesions Neutralizing Mean Mean Groups Antibody Recurrent %Lesion N = 12 Dose Titer Lesion Score Reduction Days % ReductionPhosphate- — 1:263 8.1 — 9.0 — Buffered Saline adjuvant only 50 μg × 31:331 7.1 14 8.5  6 ICP4.2 + 15 μg × 3 1:1079 4.3 47 5.1 44 adjuvant

TABLE 7 Results of therapeutic vaccination studies with ICP4.2 (SEQ IDNO: 2): viral shedding No. of animals Mean number of with no days viraldetectable viral shedding % P Groups shedding/total detected ± SEMReduction value* Phosphate- 0/11 4.5 ± 0.8 — — Buffered Saline Adjuvantonly 0/12 4.4 ± 0.7  2 0.971 ICP4.2 + 5/11 1.5 ± 0.5 67 0.004 adjuvant

TABLE 8 Results of therapeutic vaccination studies with gD2ΔTMR (SEQ IDNO: 4): lesions Mean Recurrent Mean Lesion Groups Lesion Score %Reduction Days % Reduction Adjuvant only 8.7 — 11.7 — gD2ΔTMR + 5.7 348.6 26 adjuvant

TABLE 9 Results of therapeutic vaccination studies with gD2 (SEQ ID NO:5): lesions Neutralizing Mean Mean Groups Antibody Recurrent % Lesion N= 12 Dose Titer Lesion Score Reduction Days % Reduction Phosphate- —1:263 8.1 — 9.0 — Buffered Saline Adjuvant only 50 μg × 3 1:331 7.1 148.5  6 gD2 + adjuvant 15 μg × 3 >1:6400 4.0 51 (p = 0.04) 5.0 45

TABLE 10 Results of therapeutic vaccination studies with gD2 (SEQ ID NO:5): viral shedding No. of animals Mean number with no of days viraldetectable viral shedding % P Groups shedding/total detected ± SEMReduction value* Phosphate- 0/11 4.5 ± 0.8 — — Buffered Saline Adjuvantonly 0/12 4.4 ± 0.7  2 0.971 gD2 + adjuvant 4/12 2.4 ± 0.6 47 0.047Murine Prophylactic Vaccination Protocol [Protocol B]

Female C57BL/6 mice from 6 to 8 weeks of age were immunizedsubcutaneously with antigen (HSV-2 polypeptide) plus adjuvant (12 μgdose of an ISCOM matrix with a 82:18 mixture of Quillaja saponinmatrices A and C) on day 0 and day 9. On day 11, estrous cycles weresynchronized with Depo Provera and then the mice were challenged on day16 via intravaginal deposition of 10 times the LD₅₀ of HSV-2 strain 333while under anaesthesia. All animals were monitored for morbidity(clinical score) and mortality, and body weights and vaginal swabs werecollected between days 17 and 28 post-infection. Clinical scores wererecorded using the following scale: 0=no symptoms, 1=vaginal erythema,2=vaginal erythema and edema, 3=vaginal herpetic lesions, 4=unilateralparalysis or severe genital ulceration, and 5=bilateral paralysis ordeath.

Exemplary Murine Prophylactic Vaccination Studies with VP5, gD2ΔTMR, andgD2ΔTMR Plus ICP4.2

In the experimental group, mice were immunized subcutaneously witheither 5 μg or 10 μg of antigen plus adjuvant (12 μg dose of an ISCOMmatrix with an 82:18 mixture of Quillaja saponin matrices A and C) onday 0 and day 9. Control animals received phosphate buffered saline(PBS) only, or adjuvant only.

Mice receiving PBS only or adjuvant only all died by day 9post-challenge (no survivors). In contrast, mice receiving antigenlargely survived to day 9, and 20-75% survived to day 12 post-challenge.The severity of disease symptoms (genital and neurological disease) werealso scored at either day 9 or 10 post-challenge. Mice immunized withISCOM adjuvant plus VP5, gD2ΔTMR, or gD2ΔTMR plus ICP4.2 showed asignificant decrease in disease symptoms compared to the PBS only oradjuvant only groups.

TABLE 11 Results of murine prophylactic vaccination studies Mean Disease% Score % P Survival Groups Day 10 Reduction value* Day 12 PBSonly/adjuvant only 4.81 — — 0 VP5 + adjuvant 3.13 35 0.146 38 gD2ΔTMR +adjuvant 1.44 70 0.023 75 gD2ΔTMR + 0.75 84 0.020 8 ICP4.2 + adjuvant*Student's t test relative to PBS only/adjuvant only controlResults of Murine Prophylactic Vaccination Studies with gL2s v.2, ICP4,and gD2ΔTMR

In the experimental group, C57BL/6 mice are immunized subcutaneouslywith either 5 μg or 10 μg of antigen plus adjuvant (24 μg dose of anISCOM matrix with a 91:9 mixture of Quillaja saponin matrices A and C)on day 0 and day 21. Control animals receive phosphate buffered saline(PBS) only, or adjuvant only. Animals are challenged intravaginally with10⁴ PFU HSV-2 strain 333 seven days after the last immunization. In somecases, a subset of unchallenged animals is euthanized on the day ofchallenge for immunogenicity experiments to monitor T cell and antibodyresponses to the vaccine. All challenged mice are monitored formorbidity (clinical score) and mortality, and body weights and vaginalswabs for viral shedding evaluation are collected between days 17 and 28post-infection, as described above. Clinical scores are recorded andcompared across experimental and control groups of mice.

Guinea Pig Prophylactic Vaccination Protocol [Protocol C]

Female Hartley guinea pigs from 250-350 grams (weight) were immunizedsubcutaneously with 15 μg of antigen plus adjuvant (50 μg dose of anISCOM matrix with a 91:9 mixture of Quillaja saponin matrices A and C)on day 0 and day 14-21. Sera were collected by toenail clip 2-3 weeksafter the boost and then the guinea pigs were challenged viaintravaginal deposition of 5×10⁵ PFU of HSV-2 strain MS. Vaginal-swabsamples were collected from all animals on days 30 and 32 and stored at−80° C. until assayed for virus titers by quantitative real-time PCR.Guinea pigs were evaluated daily (day 1-14), and primary genital skindisease was quantified using a lesion severity score scale from 1-4.Numerical scores were assigned to specific disease signs as follows: 0,no disease; 1, redness or swelling; 2, a few small vesicles; 3, severallarge vesicles; 4, several large vesicles with maceration. At the end ofthe study, the guinea pigs were euthanized, and the dorsal root ganglia(DRG) were harvested, stored at −80° C. until they were processed forquantitative real-time PCR analysis.

TABLE 12 Results of guinea pig prophylactic vaccination studies withgD2ΔTMR and VP5 Copies HSV-2 Viral titer, DNA/ PFU/ml Total mean acute %1 μg DRG % Groups Day 2 lesion score Reduction DNA Reduction Adjuvantonly 2.3 × 10⁶ 22.6 — 959 — gD2ΔTMR + 1.7 × 10⁶ 7.7 66% 274 71% AdjuvantVP5 + adjuvant 0.6 × 10⁶ 18.2 17% 283 70%

Vaccination studies are also performed with gL2s v.2, ICP4, and gD2ΔTMR.Female Hartley guinea pigs from 250-350 grams (weight) are immunizedsubcutaneously with 15 μg of antigen(s) plus adjuvant (50 μg dose of anISCOM matrix with a 91:9 mixture of Quillaja saponin matrix A and matrixC) on day 0 and day 21. Control animals receive phosphate bufferedsaline (PBS) only, or adjuvant only. Sera are collected by toenail clipafter immunization for monitoring total IgG responses and antibodyneutralization of virus in vitro, and the immunized guinea pigs arechallenged via intravaginal deposition of 5×10⁵ PFU of HSV-2 strain MS.Animals are monitored for acute infection for 15 days post challenge.Vaginal-swab samples are collected from all animals on days 2, 4, and 6after challenge and stored at −80° C. until assayed for virus titers byquantitative real-time PCR. Guinea pigs are evaluated daily (day 1-14),and primary genital skin disease is quantified using a lesion severityscore scale from 1-4. Numerical scores are assigned to specific diseasesigns as follows: 0, no disease; 1, redness or swelling; 2, a few smallvesicles; 3, several large vesicles; 4, several large vesicles withmaceration. After the acute phase, animals are monitored daily (day15-63) for recurrences. Groups are evaluated for length of time to firstrecurrence, monitored for genital skin disease, and in some cases,vaginal swabs taken at regular intervals to monitor viral shedding. Atthe end of the study (around D63 post challenge), the guinea pigs areeuthanized, and the dorsal root ganglia (DRG) are harvested, stored at−80° C. until they are processed for viral titers by quantitativereal-time PCR analysis.

Immunogenicity Assay I (Standard) [Protocol D]

Mice were immunized subcutaneously in the scruff of the neck with a 100μl injection of 5 μg antigen plus adjuvant (12 μg dose of an ISCOMmatrix with a 82:18 mixture of Quillaja saponin matrices A and C) insaline. The mice received one or two injections, 7 days apart. Analysisof immunogenicity occurred 7 days after the final injection.

The immunogenicity assay was an ex vivo IFN-γ ELISPOT. CD4⁺ and CD8⁺ Tcells were enriched from the spleen and separately analyzed. For theELISPOT assay, membrane plates were prepared by coating them overnightwith capture antibody and subsequently blocked by supplemented mediumfor a minimum of 2 hours at 37° C. The mice were euthanized and theirspleens harvested. The T cells were then prepared by sorting thesplenocytes for CD4⁺ and CD8⁺ T cells using magnetic beads. The blockingsolution was washed out from ELISPOT plates and the T cells were addedto the blocked plates. The plates were returned to the incubator toallow the T cells to settle. APCs were prepared by pulsing naïve Tcell-depleted splenocytes with antigen for 2 hours at 37° C. For CD4⁺ELISPOTs, APCs were pulsed with whole protein. For CD8⁺ ELISPOTs, APCswere pulsed with E. coli expressing protein plus cLLO. A medium controlwas APCs incubated for 2 hours at 37° C. with no additional antigen. Thepulsed APCs were irradiated and added to appropriate wells of platescontaining T cells. Phorbol myristate acetate (PMA) and ionomycin wereadded to separate T cell-containing control wells as a positive control.The plates were allowed to incubate for 18 hours at 37° C. under 5% CO₂.The plates were then developed using a secondary biotinylated antibody,horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC)substrate.

1. Results of Immunogenicity Assay I with ICP4.2

Immunogenicity assay I showed a robust immunogenic response for bothone- and two-injection regimens with ICP4.2. For the one-]injectionregimen, the number of IFN-γ spots per 200,000 T cells were 8 and 101for CD4⁺ and CD8⁺ T cells, respectively. For the two-injection regimen,there were 50 and 70 spots, respectively. In contrast, less than 15spots were observed for media or adjuvant alone in either CD4⁺ or CD8⁺ Tcells.

2. Results of Immunogenicity Assay I with gD2ΔTMR and gD2

Exemplary results of immunogenicity assay I are shown in FIGS. 1A and B.Robust CD4⁺ and CD8⁺ T cell responses were obtained for both full-lengthgD2 and for gD2ΔTMR. In contrast, immunization with gD2 antigentruncated immediately upstream of the transmembrane domain (denoted 306tin FIG. 1) resulted in significantly reduced responses.

Immunogenicity Assay II (Rapid) [Protocol E]

Recombinant E. coli from an HSV-2 orfeome library were induced toexpress gL2 or fragments of ICP4 protein (ICP4.2, and polypeptidesencoded by RS1.1, RS1.3.1 and RS 1.3.2). The protein was retained withinbacterial cells. The bacteria were then fixed with PFA, washedextensively with PBS and stored at −80° C. until used for immunization.

Three mice per group were immunized intraperitoneally with 1×10⁸bacteria in PBS. Mice received 1-2 additional boosters at 1 weekintervals. Seven days after the last boost, sera were collected andanalyzed in an HSV-2 neutralization assay. Five-fold serial dilutionswere prepared for plasma or serum samples in a 96-well round-bottomplate, followed by the addition of 50 PFUs HSV-2 (strain 333) to eachwell. The plates were covered and incubated at 37° C. for 1 hour. 200 μlof the virus-serum mixtures was transferred in duplicate to Vero cellsgrown in a 48-well tissue culture plate and incubated for 1 hour at 37°C. 300 μl of DMEM containing 2% FBS was then added to each well and theplates were incubated for 48 hours at 37° C. To visualize virus plaques,the plates were stained with crystal violet.

TABLE 13 Results of HSV-2 neutralization assay with gL2, ICP4.2, andICP4 polypeptides encoded by RS1.1, RS1.3.1 and RS1.3.2 HSV-2Neutralizating Antibody Immunogen Titer* E coli//gL2   1:50Ecoli//ICP4.1 <1:20 Ecoli//ICP4.2 <1:20 E. coli/ICP4.3.1    1:100 E.coli//ICP4.3.2 <1:20 Positive control    1:2500 (DL11 Mab) Negativecontrol <1:20 (naïve mouse serum) *Serum dilution that inhibits 50% ofvirus controlImmunogenicity Assay III (Overlapping Peptide Pools) [Protocol F]

Mice were immunized with 2 μg/mouse of pooled, overlapping peptides(OLP) spanning the entire sequence of gL2 and ICP4 fragments encoded byRS1.3.1 and RS1.3.2. OLPs were formulated in TiterMax adjuvant (AlexisBiochemical) in a total volume of 100 μl per mouse where adjuvantrepresented 1/3 of the subcutaneous dose. Mice were immunized on day 0,boosted on day 6 and spleens and blood were collected on day 11. Singlecell suspensions were prepared from spleens and erythrocytes were lysed.The splenocyte suspensions were then divided into halves. The first halfwas separated into APCs, CD4⁺ and CD8⁺ T cells; 200,000 T cells wereseeded per well of an IFN-γ ELISPOT plate and stimulated with 100,000APCs and OLP pool corresponding to immunization, irrelevant peptide,positive and negative control. Cells were incubated in plates overnight,after which the plates were developed and spots per well were counted.The second half of each splenocyte suspension was run as unseparatedsplenocytes (400,000/well), pulsed with peptides, and assayed asdescribed above.

Exemplary results are shown in FIGS. 2A and B as magnitude of responseper immunization group.

Vaccination with One and Two Antigens [Protocol G]

Exemplary Study 1. Immunogenicity of gD2ΔTMR and gD2ΔTMR Plus ICP4 inC57BL/6 Mice

Purified protein was mixed with adjuvant and immunized into naïve miceto evaluate the ability to make CD4⁺ and CD8⁺ T cell responses to theprotein antigens. Briefly, antigen alone (gD2ΔTMR (5 μg)) orcombinations of antigens (gD2ΔTMR and ICP4.2 (10 μg)) were mixed withadjuvant (12 μg dose of an ISCOM matrix with a 82:18 mixture of Quillajasaponin matrices A and C) and administered subcutaneously to mice,twice, 9 days apart. Seven days after the second immunization, mice wereeuthanized and spleens were harvested for ex vivo IFN-γ ELISPOT assays.CD4⁺ and CD8⁺ T cells were enriched using antibody-coated magnetic beadsand then co-cultured on IFN-γ-specific antibody-coated membranes in96-well plates. APCs were naïve splenocytes pulsed with specific ornon-specific purified proteins (as indicated) and irradiated with anx-ray irradiator. After 18 hour co-culture of T cells and APCs, capturedIFN-γ was detected with a biotinylated secondary IFN-γ-specific antibodyand visualized with horseradish peroxidase and 3-amino-9-ethylcarbazolesubstrate. Data are reported as the number of IFN-γ spot forming unitsper 2×10⁵ T cells±standard deviation of three mice per group. Asillustrated in FIGS. 3A and B, the number of IFN-γ spot forming unitsper CD4⁺ T cells or CD8⁺ T cells is increased in mice immunized withgD2ΔTMR antigen combined with ICP4.2 compared to gD2ΔTMR antigen alone.

Exemplary Study 2. Combination of gD2 and ICP4.2 Plus AdjuvantImmunization Reduced Disease Symptoms and Mortality in Mice.

The ability to trigger protective immunity after immunization with theICP4.2 protein in combination with gD2 plus adjuvant was evaluated in alethal HSV-2 challenge mouse model. Briefly, eight C57BL/6 mice pergroup were immunized with either gD2 (2 μg) or ICP4.2 (10 μg) plusadjuvant individually or with the combination of both antigens plusadjuvant. Formulations were administered subcutaneously in the scruff ofthe neck, twice, 9 days apart. Estrus cycles were synchronized with DepoProvera 5 days prior to virus infection, and animals were challengedintravaginally 7 days after the second immunization with 20 times theLD₅₀ of HSV-2 strain 333. Disease symptoms were scored post-infection,and survival monitored. Disease severity scores were as follows: 0=nosymptoms, 1=redness, 2=redness and swelling, 3=herpetic lesions,4=severe ulceration or unilateral paralysis, and 5=bilateral paralysisor death.

TABLE 14 Effect of HSV-2 proteins gD2 and ICP4.2 on disease symptoms,viral replication and mortality Mean disease Reduction % Antigen(dose){circumflex over ( )} score Reduction in P in virus Survival N = 8Day 7 disease score value** titer Day 11 PBS 3.5 ± 0.3 — — — 0% gD2* (2ug) 2.5 ± 0.2 29% 0.016 0% 25% ICP4.2 (10 ug) 1.7 ± 0.4 51% 0.005 0% 13%gD2 (2 ug) + 1.3 ± 0.3 63% 0.0004 20% 50% ICP4.2 (10 ug) {circumflexover ( )}all groups received adjuvant; *full-length protein; **Student'st test

Exemplary Study 3. Combination of gD2ΔTMR and ICP4.2 Plus AdjuvantImmunization Reduced Disease Symptoms and Mortality in Mice.

Mice were immunized with a combination of gD2ΔTMR and ICP4.2 antigensand challenged as described above. Mice immunized with the combinationof antigens plus adjuvant showed a lower mean disease score at ten daysafter virus challenge compared to animals receiving gD2ΔTMR withadjuvant.

TABLE 15 Effect of HSV-2 proteins gD2ΔTMR and gD2ΔTMR plus ICP4.2 ondisease symptoms and survival rate in mice Mean Disease % Score %Survival Groups Day 10 Reduction P value* Day 12 Adjuvant only 4.81 — —0% gD2ΔTMR + adjuvant 1.44 70 0.023 75% gD2ΔTMR + 0.75 84 0.020 88%ICP4.2 + adjuvant

Exemplary Study 4. Combination of gD2 and ICP4.2 Plus AdjuvantImmunization Reduces Severity of Recurrent Lesions when AdministeredTherapeutically to HSV-2 Infected Guinea Pigs

The ability to affect HSV-2 reactivation in infected guinea pigs aftertherapeutic immunization with antigens plus adjuvant was evaluated.Briefly, female Hartley guinea pigs were infected intravaginally with5×10⁵ pfu of HSV-2 strain MS, monitored for primary disease for 14 days,and then randomized into immunization groups (N=15). Animals wereimmunized three times subcutaneously on day 14, 21, and 35post-infection with antigen (15 μg) plus adjuvant (50 μg) or adjuvantalone, or vehicle control and scored daily for local disease severity.The scoring system was as follows: 0=no symptoms, 1=redness, 2=singlelesions, 3=large or fused lesions, 4=severe ulceration or unilateralparalysis, and 5=bilateral paralysis or death.

Table 16 shows the data as the mean recurrent lesion score for each weekafter the guinea pigs recovered from their acute disease. The guineapigs treated with a combination of gD2 and ICP4.2 antigens showed areduction in the mean lesion score at 7 (day 42) and 14 (day 49) daysafter their last immunization compared to animals receiving theindividual antigens with adjuvant.

TABLE 16 Effect of HSV-2 proteins gD2 and ICP4.2 vaccine on recurrentgenital skin disease Mean Recurrent Lesion Score Post HSV-2 InfectionAntigen + Adjuvant Day 15-21 Day 22-28 Day 29-35 Day 36-42 Day 43-49 PBS2.00 ± 0.45 1.17 ± 0.35 1.50 ± 0.50 0.87 ± 0.28 1.33 ± 0.33 gD2 1.00 ±0.30 0.67 ± 0.24 0.80 ± 0.19 0.83 ± 0.26 0.77 ± 0.28 ICP4.2 1.97 ± 0.381.07 ± 0.29 1.03 ± 0.33 0.53 ± 0.16 0.83 ± 0.29 gD2 + ICP4.2 1.43 ± 0.320.80 ± 0.27 1.07 ± 0.33 0.43 ± 0.19 0.70 ± 0.27

Exemplary Study 5. Combination of gD2ΔTMR and ICP4.2 Plus AdjuvantImmunization Reduces Severity of Recurrent Lesions when AdministeredTherapeutically to HSV-2 Infected Guinea Pigs

The ability to affect HSV-2 reactivation in infected guinea pigs aftertherapeutic immunization with antigens plus adjuvant was evaluated.Female Hartley guinea pigs were challenged intravaginally with HSV-2strain MS at 5×10⁵ pfu to establish a genital tract infection. Animalswere monitored for infection by vaginal swab on day 1 post-infection,and acute disease between days 3 and 14 post-infection. On day 14, afterresolution of primary disease, the animals were randomized into groupsof 12 and immunized subcutaneously with antigen (each HSV-2 polypeptideat 15 μg dose) plus adjuvant (50 μg dose of an ISCOM matrix with a 91:9mixture of Quillaja saponin matrices A and C). Each group received atotal of 3 vaccinations, on days 14, 21, and 35 post-infection. Genitalswabs were collected during the vaccination period to monitor viralshedding, and daily observations were recorded. Symptoms were scored ona scale from 0 to 4 based upon severity, 0=no symptoms; 1=redness orswelling; 2=a few small vesicles; 3=several large vesicles; 4=severallarge vesicles with maceration. In addition, animals with lesionsintermediate in severity between the above scores were given a score of0.5, 1.5, 2.5, or 3.5. Table 17 shows the data as the mean recurrentlesion score for each week after the guinea pigs recovered from theiracute disease. The guinea pigs treated with a combination of gD2ΔTMR andICP4.2 antigens showed a reduction in the mean lesion score after theirlast immunization compared to animals receiving the individual antigenswith adjuvant.

TABLE 17 Results of therapeutic vaccination studies with ICP4.2,gD2ΔTMR, and ICP4.2 plus gD2ΔTMR: lesions Neutralizing Mean AntibodyRecurrent % Mean % Groups Dose Titer Lesion Score Reduction Lesion DaysReduction Adjuvant only 50 μg × 3 1:250 8.9 — 10.3 — ICP4.2 + 15 μg × 31:250 6.6 26 7.7 25 adjuvant gD2ΔTMR + 15 μg × 3 1:750 7.2 20 8.3 20adjuvant ICP4.2 + 15 μg + 1:620 6.1 32 6.9 33 gD2ΔTMR + 15 μg × 3 (p =0.05) (p = 0.04) adjuvant

Exemplary Study 6. Immunogenicity of gD2ΔTMR, ICP4.2, gD2ΔTMR PlusICP4.2, gL2s v.2, UL40 Protein, and gL2s v.2 Plus UL40 Protein inC57BL/6 Mice

Purified protein was mixed with adjuvant and immunized into naïve miceto evaluate the ability to make both antibody responses and CD4⁺ andCD8⁺ T cell responses to the protein antigens. Briefly, antigen alone orcombinations of antigens were mixed with adjuvant (ISCOM matrix with a91:9 mixture of Quillaja saponin matrices A and C) and administeredsubcutaneously to mice, twice, 21 days apart. Seven days after thesecond immunization, mice were euthanized. Blood was collected todetermine antigen-specific antibody titers by direct protein ELISAassays, as described below, and spleens were harvested for ex vivo IFN-γELISPOT assays. CD4⁺ and CD8⁺ T cells were enriched usingantibody-coated magnetic beads and then co-cultured on IFN-γ-specificantibody-coated membranes in 96-well plates. APCs were naïve splenocytespulsed with specific or non-specific purified proteins (as indicated)and irradiated with an x-ray irradiator. After 18 hour co-culture of Tcells and APCs, captured IFN-γ was detected with a biotinylatedsecondary IFN-γ-specific antibody and visualized with horseradishperoxidase and 3-amino-9-ethylcarbazole substrate. Data are reported asthe number of IFN-γ spot forming units per 2×10⁵ T cells±standarddeviation of three mice per group.

Antigen-specific serum antibody titers of immunized mice were determinedby direct protein ELISA. The sera were processed from blood collectedabove and stored at −80° C. ELISA plates were coated overnight at 4° C.with 5 μg of whole protein in 0.1 M carbonate buffer, pH 9.5. Plateswere washed with TBS+0.05% Tween-20 (TBS-T) and blocked with TBS-T+1%bovine serum albumin for 1 h. Serum samples were serially diluted andincubated in the antigen-coated wells for 2 hours at room temperature.Plates were washed and probed for 1 h with goat anti-mousealkaline-phosphatase (AP)-conjugated anti-IgG at a 1:10,000 dilution.Detection of AP activity was achieved by the addition of p-Nitrophenylphosphate (pNPP; Sigmafast, Sigma-Aldrich), and the reaction stoppedwith 3N NaOH and absorbance read at 405 nm. Endpoint titers werecalculated by extrapolation of the linear portion of the serialdilutions and defining the endpoint as the dilution at which the linearportion of the curve intersects with the background cut-off. The cut-offused for data calculation was 2 times the value of the negative controlserum from a naïve mouse.

FIGS. 4A and B depict exemplary graphs illustrating the number of IFN-γspot forming units per 2×10⁵ CD4⁺ (Panel A) or CD8⁺ (Panel B) T cells,following immunization with gD2ΔTMR, ICP4.2, gD2ΔTMR plus ICP4, gL2sv.2, UL40 protein, and gL2s v.2 plus UL40 protein. Strong T cellresponses specific for ICP4.2, gD2ΔTMR, UL40 and gL2 were observed whenmice were immunized with individual antigens or in the followingcombinations: gD2ΔTMR plus ICP4.2, gL2s v.2 plus UL40 protein.

FIG. 5 illustrates IgG1 and IgG2c antibody titers against gL2s v.2, UL40protein, and gL2s v.2 plus UL40 protein. Antibodies to gL2s v.2 and UL40protein were observed when mice were immunized with cognate antigens,either individually or in combination. IgG1 and IgG2c subtypes ofantigen-specific antibodies were detected for all antigens.

Exemplary Study 7. Immunogenicity of gL2s v.2, ICP4.2, ICP4.2 Plus gL2sv.2, ICP4.9, ICP4.9 Plus gL2s v.2, ICP4.5, and ICP4.5 Plus gL2s v.2 inC57BL/6 Mice

Purified protein was mixed with adjuvant and immunized into naïve miceto evaluate the ability to make both antibody responses and CD4⁺ andCD8⁺ T cell responses to the protein antigens as described for ExemplaryStudy 6 above, except that APCs were pulsed with either the indicatedpurified proteins, or with pools of overlapping peptides spanning theindicated proteins. Mice received gL2s v.2, ICP4.5 at two differentdoses, ICP4.9 at two different doses, ICP4.2, or combinations of ICP4.5,ICP4.9, and ICP4.2 plus gL2s v.2.

FIGS. 6A and B depict exemplary graphs illustrating the average numberof IFN-γ spot forming units per 2×10⁵ CD4⁺ (Panel A) or CD8⁺ (Panel B) Tcells, following immunization with gL2s v.2, ICP4.2, ICP4.2 plus gL2sv.2, ICP4.9, ICP4.9 plus gL2s v.2, ICP4.5, and ICP4.5 plus gL2s v.2.Strong gL2s v.2, ICP4.2, ICP4.9, and ICP4.5 T cell responses wereobtained. Some expected cross-reactivity due to sequence overlap amongproteins was observed. Unexpected cross-reactivity was observed betweengL2s v.2 and ICP4.5.

FIGS. 6C and D depict exemplary graphs illustrating the average numberof IFN-γ spot forming units per 2×10⁵ CD4⁺ (Panel C) or CD8⁺ (Panel D) Tcells, following immunization with gL2s v.2, ICP4.2, ICP4.2 plus gL2sv.2, ICP4.9, ICP4.9 plus gL2s v.2, ICP4.5, and ICP4.5 plus gL2s v.2, asin FIGS. 6A and B, except that APCs were pulsed with pools ofoverlapping peptides spanning the indicated proteins rather than withpurified proteins.

FIG. 7 depicts an exemplary graph illustrating antibody titers againstgL2s v.2, ICP4.5, gL2s v.2 plus ICP4.5, ICP4.9, gL2s v.2 plus ICP4.9,ICP4.2, and gL2s v2 plus ICP4.2. Mice mounted strong antibody responsesto ICP4.2, ICP4.9, and ICP4.5. Weak gL2s v.2-specific antibody responseswere observed when this antigen was paired with each of the ICP4fragments.

Example 3 In Vivo Data: Murine Prophylactic Vaccination Studies withProtein Antigen Plus DNA, and DNA Only

Exemplary Studies 1 and 2. Immunization with gL2s v.2 Protein, pUs6 DNA(Encoding gD2), and pUL1 DNA (Encoding gL2) with gL2s v.2 Protein BoostReduced Disease Symptoms and Mortality in C57BL/6 Mice

In these studies, groups of C57BL/6 mice were immunized intramuscularlyon day 0 and 21 with 10 μg of gL2s v.2 protein plus adjuvant (24 μg doseof an ISCOM matrix with a 91:9 mixture of Quillaja saponin matrix A andmatrix C), or 100 μg of DNA plasmid encoding gD2 (pUs6) combined with 50μg of DNA plasmid encoding murine IL-12 (pIL-12) as adjuvant. Anothergroup received 100 μg of DNA plasmid encoding gL2 (pUL1) and 50 μg ofpIL-12 as the prime immunization at day 0, and 10 μg of gL2s v.2 proteinplus ISCOM adjuvant for the boost on day 21. Control animals received 50μg of pIL-12 or ISCOM adjuvant only. Animals were challengedintravaginally with 10⁴ PFU HSV-2 strain 333 seven days after the lastimmunization. In some cases, a subset of unchallenged animals wereeuthanized on the day of challenge for immunogenicity experiments tomonitor T cell and antibody responses to the vaccine (as described inExemplary Studies 3 and 4 below). All challenged mice were monitored formorbidity (clinical score) and mortality, and body weights and vaginalswabs for viral shedding evaluation are collected between days 1 and 10post-infection, as described above. Clinical scores were recorded andcompared across experimental and control groups of mice.

Table 18 and Table 19 illustrate exemplary results of two separateprophylactic vaccination studies.

TABLE 18 Vaccination study 1: mouse protection results for gL2s v.2,pUs6 DNA, and pUL1 DNA with gL2s v.2 protein boost Mean Disease Score %% Survival Groups (N = 8) Day 14 Reduction P value* Day 14 PBS +adjuvant 4.75 — — 13 gL2s v.2 + 3.38 29 0.093 63 adjuvant pUs6 + pIL122.00 58 0.007 88 pUL1 + 2.50 47 0.047 63 pIL12/gL2s v.2 + adjuvant*one-sided Student's t test

TABLE 19 Vaccination study 2: mouse protection results for gL2s v.2,pUs6 DNA, and pUL1 DNA plus gL2s v.2 boost Mean Disease % SurvivalGroups (N = 8) Score Day 14 % Reduction P value* Day 14 PBS + adjuvant4.31 — — 25 gL2s v.2 + 3.44 20 0.238 50 adjuvant pIL12 4.75 — — 25pUs6 + pIL12 2.13 55 0.010 88 pUL1+ pIL12/ 2.94 38 0.075 63 gL2s v.2 +adjuvant *one-sided Student's t test

Exemplary Study 3. Immunogenicity of pRS1 DNA (Encoding ICP4); pUL1 DNA(Encoding gL2); pRS1 DNA (Encoding ICP4) Plus pUL1 DNA (Encoding gL2);gL2 Protein; and pUL1 DNA (Encoding gL2) with gL2s v.2 Protein Boost inC57BL/6 Mice

C57BL/6 mice were immunized with plasmid DNA encoding gD2, ICP4 or gL2along with plasmid DNA encoding IL-12 or gL2s v.2 protein with orwithout ISCOM adjuvant, as described in Exemplary Studies 1 and 2 above.Two groups were primed with plasmid DNA encoding gL2 and plasmid DNAencoding IL12 and boosted with gL2s v.2 protein. In addition, mice wereimmunized with a combination of gL2s v.2 protein and ICP4 plasmid DNAalong with IL12 plasmid. Seven days post last immunizations, blood wascollected to determine antigen-specific antibody titers by directprotein ELISA assays, as described below, and spleens were harvested forex vivo IFN-γ ELISPOT assays. CD4⁺ and CD8⁺ T cells were enriched usingantibody-coated magnetic beads and then co-cultured on IFN-γ-specificantibody-coated membranes in 96-well plates. APCs were naïve splenocytespulsed with pools of overlapping peptides spanning indicated proteinsand irradiated with an x-ray irradiator. After 18 hour co-culture of Tcells and APCs, captured IFN-γ was detected with a biotinylatedsecondary IFN-γ-specific antibody and visualized with horseradishperoxidase and 3-amino-9-ethylcarbazole substrate. Data are reported asthe number of IFN-γ spot forming units per 2×10⁵ T cells±standarddeviation of three mice per group.

Antigen-specific serum antibody titers of immunized mice were determinedby direct protein ELISA. The sera were processed from blood collectedabove and stored at −80° C. ELISA plates were coated overnight at 4° C.with 5 μg of whole protein in 0.1 M carbonate buffer, pH 9.5. Plateswere washed with TBS+0.05% Tween-20 (TBS-T) and blocked with TBS-T+1%bovine serum albumin for 1 h. Serum samples were serially diluted andincubated in the antigen-coated wells for 2 hours at room temperature.Plates were washed and probed for 1 h with goat anti-mousealkaline-phosphatase (AP)-conjugated anti-IgG at a 1:10,000 dilution.Detection of AP activity was achieved by the addition of p-Nitrophenylphosphate (pNPP; Sigmafast, Sigma-Aldrich), and the reaction stoppedwith 3N NaOH and absorbance read at 405 nm. Endpoint titers werecalculated by extrapolation of the linear portion of the serialdilutions and defining the endpoint as the dilution at which the linearportion of the curve intersects with the background cut-off. The cut-offused for data calculation was 2 times the value of the negative controlserum from a naïve mouse.

FIGS. 8A and B depict exemplary graphs illustrating the average numberof IFN-γ spot forming units per 2×10⁵ CD4⁺ (Panel A) or CD8⁺ (Panel B) Tcells, following immunization with pRS1 DNA (encoding ICP4); pUL1 DNA(encoding gL2); pRS1 DNA (encoding ICP4) plus pUL1 DNA (encoding gL2);pUL1 DNA (encoding gL2) with gL2s v.2 protein boost; and gL2s v.2protein. Strong gL2- or gL2s v.2-specific T cell response was observedin mice that received gL2s v.2 protein with ISCOM adjuvant for prime andboost immunizations. Even stronger response was observed in mice thatwere primed with pUL1 and pIL-12 DNA and boosted with gL2s v.2 protein.Weak T cell response was observed when pUL1 DNA was used for priming andboost. Very low levels of ICP4-specific T cells were detected.

FIG. 9 depicts an exemplary graph illustrating total IgG antibody titersagainst ICP4.2 and gL2s v.2 (also gD2ΔTMR). Prime and/or boost with gL2sv.2 protein was required for development of gL2s v.2-specificantibodies.

Exemplary Study 4. Immunogenicity of gL2s v.2 Protein; pUL1 DNA(Encoding gL2); pUL1 DNA (Encoding gL2) with gL2s v.2 Protein Boost;pRS1 DNA (Encoding ICP4); pRS1 DNA (Encoding ICP4) Plus pUL1 DNA(Encoding gL2); and pUs6 DNA (Encoding gD2) in C57BL/6 Mice

C57BL/6 mice were immunized as described in Exemplary Studies 1 and 2above with gL2s v.2 protein plus adjuvant; pUL1 DNA (encoding gL2); pUL1DNA (encoding gL2) with gL2s v.2 protein plus adjuvant boost; pRS1 DNA(encoding ICP4); pRS1 DNA (encoding ICP4) plus pUL1 DNA (encoding gL2);and pUs6 DNA (encoding gD2). Seven days post last immunization, spleenswere harvested for ex vivo IFN-γ ELISPOT assays as described inExemplary Study 3 above.

FIGS. 10A and B depict exemplary graphs illustrating the number of IFN-γspot forming units per 2×10⁵ CD4⁺ (Panel A) or CD8⁺ (Panel B) T cells,following immunization with gL2s v.2 protein; pUL1 DNA (encoding gL2);pUL1 DNA (encoding gL2) with gL2s v.2 protein boost; pRS1 DNA (encodingICP4); pRS1 DNA (encoding ICP4) plus pUL1 DNA (encoding gL2); and pUs6DNA (encoding gD2). Strong gL2- or gL2s v.2-specific T cell response wasobserved in mice that received gL2s v.2 protein with ISCOM adjuvant ormice that received pUL1 and pIL12 DNA plasmid for prime and boostimmunizations. The response was augmented further in mice that wereprimed with pUL1 and pIL-12 DNA plasmids, then boosted with gL2s v.2protein plus ISCOM adjuvant.

Exemplary Study 5. Immunogenicity of pRS1 DNA (Encoding ICP4), pRS1.9DNA (Encoding ICP4.9), and pUs4 DNA (Encoding gG2) with CorrespondingDNA Boost; pRS1 DNA (Encoding ICP4), pRS1.9 DNA (Encoding ICP4.9), andpUs4 DNA (Encoding gG2) with ICP4.2 Boost

C57BL/6 mice were immunized as described in Exemplary Studies 1 and 2above with plasmid DNA encoding ICP4, ICP4.9, gG2 or empty plasmid incombination with a plasmid encoding mouse IL-12. Mice were then dividedinto three groups. Mice of the first group were boosted on day 21 thesame way they were primed. Mice of the second group were boosted on day21 with ICP4.2 protein plus ISCOM adjuvant. Mice of the third group wereboosted on days 21 and 35 the same way they were primed. Seven days postlast immunization, spleens were harvested for ex vivo IFN-γ ELISPOTassays as described in Exemplary Study 3 above.

FIG. 11 depicts an exemplary graph illustrating the number of IFN-γ spotforming units per 2×10⁵ CD4⁺ (left panel) or CD8⁺ (right panel) T cells,following immunization with pRS1 DNA (encoding ICP4), pRS1.9 DNA(encoding ICP4.9), and pUs4 DNA (encoding gG2) with corresponding DNAboost (first group of mice) or with ICP4.2 protein boost (second groupof mice) on day 21. Strong ICP4.2-specific T cell response was observedin mice primed with pRS1 or pRS1.9 DNA, then boosted with ICP4.2 proteinplus ISCOM adjuvant.

FIG. 12 depicts an exemplary graph illustrating the number of IFN-γ spotforming units per 2×10⁵ CD4⁺ (left panel) or CD8⁺ (right panel) T cells,following immunization with pRS1 DNA (encoding ICP4) and pUs4 DNA(encoding gG2) with corresponding DNA boosts on days 21 and 35 (thirdgroup of mice).

SEQUENCES SEQ ID NO: 1 = ICP4SAEQRKKKKTTTTTQGRGAEVAMADEDGGRLRAAAETTGGPGSPDPADGPPPTPNPDRRPAARPGFGWHGGPEENEDEADDAAADADADEAAPASGEAVDEPAADGVVSPRQLALLASMVDEAVRTIPSPPPERDGAQEEAARSPSPPRTPSMRADYGEENDDDDDDDDDDDRDAGRWVRGPETTSAVRGAYPDPMASLSPRPPAPRRHHHHHHHRRRRAPRRRSAASDSSKSGSSSSASSASSSASSSSSASASSSDDDDDDDAARAPASAADHAAGGTLGADDEEAGVPARAPGAAPRPSPPRAEPAPARTPAATAGRLERRRARAAVAGRDATGRFTAGRPRRVELDADAASGAFYARYRDGYVSGEPWPGAGPPPPGRVLYGGLGDSRPGLWGAPEAEEARARFEASGAPAPVWAPELGDAAQQYALITRLLYTPDAEAMGWLQNPRVAPGDVALDQACFRISGAARNSSSFISGSVARAVPHLGYAMAAGRFGWGLAHVAAAVAMSRRYDRAQKGFLLTSLRRAYAPLLARENAALTGARTPDDGGDANRHDGDDARGKPAAAAAPLPSAAASPADERAVPAGYGAAGVLAALGRLSAAPASAPAGADDDDDDDGAGGGGGGRRAEAGRVAVECLAACRGILEALAEGFDGDLAAVPGLAGARPAAPPRPGPAGAAAPPHADAPRLRAWLRELRFVRDALVLMRLRGDLRVAGGSEAAVAAVRAVSLVAGALGPALPRSPRLLSSAAAAAADLLFQNQSLRPLLADTVAAADSLAAPASAPREARKRKSPAPARAPPGGAPRPPKKSRADAPRPAAAPPAGAAPPAPPTPPPRPPRPAALTRRPAEGPDPQGGWRRQPPGPSHTPAPSAAALEAYCAPRAVAELTDHPLFPAPWRPALMFDPRALASLAARCAAPPPGGAPAAFGPLRASGPLRRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGAGDAMAPGAPDFCEDEAHSHRACARWGLGAPLRPVYVALGRDAVRGGPAELRGPRREFCARALLEPDGDAPPLVLRDDADAGPPPQIRWASAAGRAGTVLAAAGGGVEVVGTAAGLATPPRREPVDMDAELEDDDDGLFGE SEQ ID NO: 2 = ICP4 internal fragmentMVLYGGLGDSRPGLWGAPEAEEARARFEASGAPAPVWAPELGDAAQQYALITRLLYTPDAEAMGWLQNPRVAPGDVALDQACFRISGAARNSSSFISGSVARAVPHLGYAMAAGRFGWGLAHVAAAVAMSRRYDRAQKGFLLTSLRRAYAPLLARENAALTGARTPDDGGDANRRDGDDARGKPAAAAAPLPSAAASPADERAVPAGYGAAGVLAALGRLSAAPASAPAGADDDDDDDDGAGGGGGGGGGGGGRRAEAGRVAVECLAACRGILEALAEGFDGDLAAVPGLAGARPAAPPRPGPAGAAAPPHADAPRLRAWLRELRFVRDALVLMRLRGDLRVAGGSEAAVAAVRAVSLVAGALGPALPRSPRLLSSAAAAAADLLFQNQSL SEQ ID NO: 3 = gL2MGFVCLFGLVVMGAWGAWGGSQATEYVLRSVIAKEVGDILRVPCMRTPADDVSWRYEAPSVIDYARIDGIFLRYHCPGLDTFLWDRHAQRAYLVNPFLFAAGFLEDLSHSVFPADTQETTTRRALYKEIRDALGSRKQAVSHAPVRAGCVNFDYSRTRRCVGRRDLRPANTTSTWEPPVSSDDEASSQSKPLATQPPVLALSNAPPRRVSPTRGRRRHTRLRRNSEQ ID NO: 4 =gD2 internal deletion dD2ΔTMR encoded by construct US6ΔTMRNRWKYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY SEQ ID NO: 5 =predicted sequence for gD2 encoded by US6MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGALAGSTLAVLVIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY SEQ ID NO: 6 = ICP34.5 encoded by RL1MSRRRGPRRRGPRRRPRPGAPAVPRPGAPAVPRPGALPTADSQMVPAYDSGTAVESAPAASSLLRRWLLVPQADDSDDADYAGNDDAEWANSPPSEGGGKAPEAPHAAPAAACPPPPPRKERGPQRPLPPHLALRLRTTTEYLARLSLRRRRPPASPPADAPRGKVCFSPRVQVRHLVAWETAARLARRGSWARERADRDRFRRRVAAAEAVIGPCLEPEARARARARARAHEDGGPAEEEEAAAAARGSSAAAGPGRRAV SEQ ID NO: 7 = ICP0 encoded by RL2MEPRPGTSSRADPGPERPPRQTPGTQPAAPHAWGMLNDMQWLASSDSEEETEVGISDDDLHRDSTSEAGSTDTEMFEAGLMDAATPPARPPAERQGSPTPADAQGSCGGGPVGEEEAEAGGGGDVCAVCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDPRTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSLGGHTVRALSPTPPWPGTDDEDDDLADVDYVPPAPRRAPRRGGGGAGATRGTSQPAATRPAPPGAPRSSSSGGAPLRAGVGSGSGGGPAVAAVVPRVASLPPAAGGGRAQARRVGEDAAAAEGRTPPARQPRAAQEPPIVISDSPPPSPRRPAGPGPLSFVSSSSAQVSSGPGGGGLPQSSGRAARPRAAVAPRVRSPPRAAAAPVVSASADAAGPAPPAVPVDAHRAPRSRMTQAQTDTQAQSLGRAGATDARGSGGPGAEGGPGVPRGTNTPGAAPHAAEGAAARPRKRRGSDSGPAASSSASSSAAPRSPLAPQGVGAKRAAPRRAPDSDSGDRGHGPLAPASAGAAPPSASPSSQAAVAAASSSSASSSSASSSSASSSSASSSSASSSSASSSSASSSAGGAGGSVASASGAGERRETSLGPRAAAPRGPRKCARKTRHAEGGPEPGARDPAPGLTRYLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGALDFHGLRSRHPWSREQGAPAPAGDAPAGHGE SEQ ID NO: 8 =ICP4 internal fragment encoded by construct RS1.1 (#1-400)MSAEQRKKKKTTTTTQGRGAEVAMADEDGGRLRAAAETTGGPGSPDPADGPPPTPNPDRRPAARPGFGWHGGPEENEDEADDAAADADADEAAPASGEAVDEPAADGVVSPRQLALLASMVDEAVRTIPSPPPERDGAQEEAARSPSPPRTPSMRADYGEENDDDDDDDDDDDRDAGRWVRGPETTSAVRGAYPDPMASLSPRPPAPRRHHHHHHHRRRRAPRRRSAASDSSKSGSSSSASSASSSASSSSSASASSSDDDDDDDAARAPASAADHAAGGTLGADDEEAGVPARAPGAAPRPSPPRAEPAPARTPAATAGRLERRRARAAVAGRDATGRFTAGRPRRVELDADAASGAFYARYRDGYVSGEPWPGAGPPPPGRVLYGGLGDSRPGLWGAP SEQ ID NO: 9 =ICP4 internal fragment encoded by construct RS1.3.1 (#750-1024)SSAAAAAADLLFQNQSLRPLLADTVAAADSLAAPASAPREARKRKSPAPARAPPGGAPRPPKKSRADAPRPAAAPPAGAAPPAPPTPPPRPPRPAALTRRPAEGPDPQGGWRRQPPGPSHTPAPSAAALEAYCAPRAVAELTDHPLFPAPWRPALMFDPRALASLAARCAAPPPGGAPAAFGPLRASGPLRRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQ SEQ ID NO: 10 =ICP4 internal fragment encoded by construct RS1.3.2 (#1008-1319)WAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGAGDAMAPGAPDFCEDEAHSHRACARWGLGAPLRPVYVALGRDAVRGGPAELRGPRREFCARALLEPDGDAPPLVLRDDADAGPPPQIRWASAAGRAGTVLAAAGGGVEVVGTAAGLATPPRREPVDMDAELEDDDDGLFGE SEQ ID NO: 11 =ICP4 internal fragment encoded by construct RS1.3 (#750-1319)SSAAAAAADLLFQNQSLRPLLADTVAAADSLAAPASAPREARKRKSPAPARAPPGGAPRPPKKSRADAPRPAAAPPAGAAPPAPPTPPPRPPRPAALTRRPAEGPDPQGGWRRQPPGPSHTPAPSAAALEAYCAPRAVAELTDHPLFPAPWRPALMFDPRALASLAARCAAPPPGGAPAAFGPLRASGPLRRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGAGDAMAPGAPDFCEDEAHSHRACARWGLGAPLRPVYVALGRDAVRGGPAELRGPRREFCARALLEPDGDAPPLVLRDDADAGPPPQIRWASAAGRAGTVLAAAGGGVEVVGTAAGLATPPRREPVDMDAELEDDDDGLFGE SEQ ID NO: 12 =ICP4 internal fragment encoded by construct RS1.4 (#340-883)TAGRPRRVELDADAASGAFYARYRDGYVSGEPWPGAGPPPPGRVLYGGLGDSRPGLWGAPEAEEARARFEASGAPAPVWAPELGDAAQQYALITRLLYTPDAEAMGWLQNPRVAPGDVALDQACFRISGAARNSSSFISGSVARAVPHLGYAMAAGRFGWGLAHVAAAVAMSRRYDRAQKGFLLTSLRRAYAPLLARENAALTGARTPDDGGDANRHDGDDARGKPAAAAAPLPSAAASPADERAVPAGYGAAGVLAALGRLSAAPASAPAGADDDDDDDGAGGGGGGRRAEAGRVAVECLAACRGILEALAEGFDGDLAAVPGLAGARPAAPPRPGPAGAAAPPHADAPRLRAWLRELRFVRDALVLMRLRGDLRVAGGSEAAVAAVRAVSLVAGALGPALPRSPRLLSSAAAAAADLLFQNQSLRPLLADTVAAADSLAAPASAPREARKRKSPAPARAPPGGAPRPPKKSRADAPRPAAAPPAGAAPPAPPTPPPRPPRPAALTRRPAEGPDPQGGWRRQPPGPSHTPAPSAAALEAYCA SEQ ID NO: 13 =ICP4 internal fragment encoded by construct RS1.5 (#775-1318)AAADSLAAPASAPREARKRKSPAPARAPPGGAPRPPKKSRADAPRPAAAPPAGAAPPAPPTPPPRPPRPAALTRRPAEGPDPQGGWRRQPPGPSHTPAPSAAALEAYCAPRAVAELTDHPLFPAPWRPALMFDPRALASLAARCAAPPPGGAPAAFGPLRASGPLRRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGAGDAMAPGAPDFCEDEAHSHRACARWGLGAPLRPVYVALGRDAVRGGPAELRGPRREFCARALLEPDGDAPPLVLRDDADAGPPPQIRWASAAGRAGTVLAAAGGGVEVVGTAAGLATPPRREPVDMDAELEDDDDGLFGE SEQ ID NO: 14 =ICP4 internal fragment encoded by construct RS1.6 (#210-1318)HHHHHHHRRRRAPRRRSAASDSSKSGSSSSASSASSSASSSSSASASSSDDDDDDDAARAPASAADHAAGGTLGADDEEAGVPARAPGAAPRPSPPRAEPAPARTPAATAGRLERRRARAAVAGRDATGRFTAGRPRRVELDADAASGAFYARYRDGYVSGEPWPGAGPPPPGRVLYGGLGDSRPGLWGAPEAEEARARFEASGAPAPVWAPELGDAAQQYALITRLLYTPDAEAMGWLQNPRVAPGDVALDQACFRISGAARNSSSFISGSVARAVPHLGYAMAAGRFGWGLAHVAAAVAMSRRYDRAQKGFLLTSLRRAYAPLLARENAALTGARTPDDGGDANRHDGDDARGKPAAAAAPLPSAAASPADERAVPAGYGAAGVLAALGRLSAAPASAPAGADDDDDDDGAGGGGGGRRAEAGRVAVECLAACRGILEALAEGFDGDLAAVPGLAGARPAAPPRPGPAGAAAPPHADAPRLRAWLRELRFVRDALVLMRLRGDLRVAGGSEAAVAAVRAVSLVAGALGPALPRSPRLLSSAAAAAADLLFQNQSLRPLLADTVAAADSLAAPASAPREARKRKSPAPARAPPGGAPRPPKKSRADAPRPAAAPPAGAAPPAPPTPPPRPPRPAALTRRPAEGPDPQGGWRRQPPGPSHTPAPSAAALEAYCAPRAVAELTDHPLFPAPWRPALMFDPRALASLAARCAAPPPGGAPAAFGPLRASGPLRRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGAGDAMAPGAPDFCEDEAHSHRACARWGLGAPLRPVYVALGRDAVRGGPAELRGPRREFCARALLEPDGDAPPLVLRDDADAGPPPQIRWASAAGRAGTVLAAAGGGVEVVGTAAGLATPPRREPVDMDAELEDDDDGLFGE SEQ ID NO: 15 =ICP4 internal fragment encoded by construct RS1.7 (deletion of #391-544)MSAEQRKKKKTTTTTQGRGAEVAMADEDGGRLRAAAETTGGPGSPDPADGPPPTPNPDRRPAARPGFGWHGGPEENEDEADDAAADADADEAAPASGEAVDEPAADGVVSPRQLALLASMVDEAVRTIPSPPPERDGAQEEAARSPSPPRTPSMRADYGEENDDDDDDDDDDDRDAGRWVRGPETTSAVRGAYPDPMASLSPRPPAPRRHHHHHHHRRRRAPRRRSAASDSSKSGSSSSASSASSSASSSSSASASSSDDDDDDDAARAPASAADHAAGGTLGADDEEAGVPARAPGAAPRPSPPRAEPAPARTPAATAGRLERRRARAAVAGRDATGRFTAGRPRRVELDADAASGAFYARYRDGYVSGEPWPGAGPPPPGRVLYGGLGARTPDDGGDANRHDGDDARGKPAAAAAPLPSAAASPADERAVPAGYGAAGVLAALGRLSAAPASAPAGADDDDDDDGAGGGGGGRRAEAGRVAVECLAACRGILEALAEGFDGDLAAVPGLAGARPAAPPRPGPAGAAAPPHADAPRLRAWLRELRFVRDALVLMRLRGDLRVAGGSEAAVAAVRAVSLVAGALGPALPRSPRLLSSAAAAAADLLFQNQSLRPLLADTVAAADSLAAPASAPREARKRKSPAPARAPPGGAPRPPKKSRADAPRPAAAPPAGAAPPAPPTPPPRPPRPAALTRRPAEGPDPQGGWRRQPPGPSHTPAPSAAALEAYCAPRAVAELTDHPLFPAPWRPALMFDPRALASLAARCAAPPPGGAPAAFGPLRASGPLRRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGAGDAMAPGAPDFCEDEAHSHRACARWGLGAPLRPVYVALGRDAVRGGPAELRGPRREFCARALLEPDGDAPPLVLRDDADAGPPPQIRWASAAGRAGTVLAAAGGGVEVVGTAAGLATPPRREPVDMDAELEDDDDGLFGE SEQ ID NO: 16 =ICP4 internal fragment encoded by construct RS1.8 (deletion of #786-868)MSAEQRKKKKTTTTTQGRGAEVAMADEDGGRLRAAAETTGGPGSPDPADGPPPTPNPDRRPAARPGFGWHGGPEENEDEADDAAADADADEAAPASGEAVDEPAADGVVSPRQLALLASMVDEAVRTIPSPPPERDGAQEEAARSPSPPRTPSMRADYGEENDDDDDDDDDDDRDAGRWVRGPETTSAVRGAYPDPMASLSPRPPAPRRHHHHHHHRRRRAPRRRSAASDSSKSGSSSSASSASSSASSSSSASASSSDDDDDDDAARAPASAADHAAGGTLGADDEEAGVPARAPGAAPRPSPPRAEPAPARTPAATAGRLERRRARAAVAGRDATGRFTAGRPRRVELDADAASGAFYARYRDGYVSGEPWPGAGPPPPGRVLYGGLGDSRPGLWGAPEAEEARARFEASGAPAPVWAPELGDAAQQYALITRLLYTPDAEAMGWLQNPRVAPGDVALDQACFRISGAARNSSSFISGSVARAVPHLGYAMAAGRFGWGLAHVAAAVAMSRRYDRAQKGFLLTSLRRAYAPLLARENAALTGARTPDDGGDANRHDGDDARGKPAAAAAPLPSAAASPADERAVPAGYGAAGVLAALGRLSAAPASAPAGADDDDDDDGAGGGGGGRRAEAGRVAVECLAACRGILEALAEGFDGDLAAVPGLAGARPAAPPRPGPAGAAAPPHADAPRLRAWLRELRFVRDALVLMRLRGDLRVAGGSEAAVAAVRAVSLVAGALGPALPRSPRLLSSAAAAAADLLFQNQSLRPLLADTVAAADSLAAPASTPAPSAAALEAYCAPRAVAELTDHPLFPAPWRPALMFDPRALASLAARCAAPPPGGAPAAFGPLRASGPLRRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGAGDAMAPGAPDFCEDEAHSHRACARWGLGAPLRPVYVALGRDAVRGGPAELRGPRREFCARALLEPDGDAPPLVLRDDADAGPPPQIRWASAAGRAGTVLAAAGGGVEVVGTAAGLATPPRREPVDMDAELEDDDDGLFGE SEQ ID NO: 17 =predicted sequence for uracil DNA glycosylase encoded by UL2MFSASTTPEQPLGLSGDATPPLPTSVPLDWAAFRRAFLIDDAWRPLLEPELANPLTARLLAEYDRRCQTEEVLPPREDVFSWTRYCTPDDVRVVIIGQDPYHHPGQAHGLAFSVRADVPVPPSLRNVLAAVKNCYPDARMSGRGCLEKWARDGVLLLNTTLTVKRGAAASHSKLGWDRFVGGVVQRLAARRPGLVFMLWGAHAQNAIRPDPRQHYVLKFSHPSPLSKVPFGTCQHFLAANRYLETRDIMPIDWSV SEQ ID NO: 18 =predicted sequence for tegument protein encoded by UL11MGLAFSGARPCCCRHNVITTDGGEVVSLTAHEFDVVDIESEEEGNFYVPPDVRVVTRAPGPQYRRASDPPSRHTRRRDPDVARPPATLTPPLSDSE SEQ ID NO: 19 =gL2 secreted v.1 encoded by construct UL1s v.1NRWGFVCLFGLVVMGAWGAWGGSQATEYVLRSVIAKEVGDILRVPCMRTPADDVSWRYEAPSVIDYARIDGIFLRYHCPGLDTFLWDRHAQRAYLVNPFLFAAGFLEDLSHSVFPADTQETTTRRALYKEIRDALGSRKQAVSHAPVRAGCVNFDYSRTRRCVGRRDLRPANTTSTWEPPVSSDDEASSQSKPLATQPPVLALSNAPPRRVSPTRGRRRHTRLRRNSEQ ID NO: 20 = predicted sequence for VP5 encoded by construct UL19aDYDIPTTENLYFQGMAAPARDPPGYRYAAAMVPTGSILSTIEVASHRRLFDFFARVRSDENSLYDVEFDALLGSYCNTLSLVRFLELGLSVACVCTKFPELAYMNEGRVQFEVHQPLIARDGPHPVEQPVHNYMTKVIDRRALNAAFSLATEAIALLTGEALDGTGISLHRQLRAIQQLARNVQAVLGAFERGTADQMLHVLLEKAPPLALLLPMQRYLDNGRLATRVARATLVAELKRSFCDTSFFLGKAGHRREAIEAWLVDLTTATQPSVAVPRLTHADTRGRPVDGVLVTTAAIKQRLLQSFLKVEDTEADVPVTYGEMVLNGANLVTALVMGKAVRSLDDVGRHLLEMQEEQLEANRETLDELESAPQTTRVRADLVAIGDRLVFLEALEKRIYAATNVPYPLVGAMDLTFVLPLGLFNPAMERFAAHAGDLVPAPGHPEPRAFPPRQLFFWGKDHQVLRLSMENAVGTVCHPSLMNIDAAVGGVNHDPVEAANPYGAYVAAPAGPGADMQQRFLNAWRQRLAHGRVRWVAECQMTAEQFMQPDNANLALELHPAFDFFAGVADVELPGGEVPPAGPGAIQATWRVVNGNLPLALCPVAFRDARGLELGVGRHAMAPATIAAVRGAFEDRSYPAVFYLLQAAIHGSEHVFCALARLVTQCITSYWNNTRCAAFVNDYSLVSYIVTYLGGDLPEECMAVYRDLVAHVEALAQLVDDFTLPGPELGGQAQAELNHLMRDPALLPPLVWDCDGLMRHAALDRHRDCRIDAGEHEPVYAAACNVATADFNRNDGRLLHNTQARAADAADDRPHRPADWTVHHKIYYYVLVPAFSRGRCCTAGVRFDRVYATLQNMVVPEIAPGEECPSDPVTDPAHPLHPANLVANTVNAMFHNGRVVVDGPAMLTLQVLAHNMAERTTALLCSAAPDAGANTASTANMRIFDGALHAGVLLMAPQHLDHTIQNGEYFYVLPVHALFAGADHVANAPNFPPALRDLARHVPLVPPALGANYFSSIRQPVVQHARESAAGENALTYALMAGYFKMSPVALYHQLKTGLHPGFGFTVVRQDRFVTENVLFSERASEAYFLGQLQVARHETGGGVSFTLTQPRGNVDLGVGYTAVAATATVRNPVTDMGNLPQNFYLGRGAPPLLDNAAAVYLRNAVVAGNRLGPAQPLPVFGCAQVPRRAGMDHGQDAVCEFIATPVATDINYFRRPCNPRGRAAGGVYAGDKEGDVIALMYDHGQSDPARPFAATANPWASQRFSYGDLLYNGAYHLNGASPVLSPCFKFFTAADITAKHRCLERLIVETGSAVSTATAASDVQFKRPPGCRELVEDPCGLFQEAYPITCASDPALLRSARDGEAHARETHFTQYLIYDASPLKGLSL SEQ ID NO: 21 = VP5 encoded by construct UL19ΔTEVMAAPARDPPGYRYAAAMVPTGSILSTIEVASHRRLFDFFARVRSDENSLYDVEFDALLGSYCNTLSLVRFLELGLSVACVCTKFPELAYMNEGRVQFEVHQPLIARDGPHPVEQPVHNYMTKVIDRRALNAAFSLATEAIALLTGEALDGTGISLHRQLRAIQQLARNVQAVLGAFERGTADQMLHVLLEKAPPLALLLPMQRYLDNGRLATRVARATLVAELKRSFCDTSFFLGKAGHRREAIEAWLVDLTTATQPSVAVPRLTHADTRGRPVDGVLVTTAAIKQRLLQSFLKVEDTEADVPVTYGEMVLNGANLVTALVMGKAVRSLDDVGRHLLEMQEEQLEANRETLDELESAPQTTRVRADLVAIGDRLVFLEALEKRIYAATNVPYPLVGAMDLTFVLPLGLFNPAMERFAAHAGDLVPAPGHPEPRAFPPRQLFFWGKDHQVLRLSMENAVGTVCHPSLMNIDAAVGGVNHDPVEAANPYGAYVAAPAGPGADMQQRFLNAWRQRLAHGRVRWVAECQMTAEQFMQPDNANLALELHPAFDFFAGVADVELPGGEVPPAGPGAIQATWRVVNGNLPLALCPVAFRDARGLELGVGRHAMAPATIAAVRGAFEDRSYPAVFYLLQAAIHGSEHVFCALARLVTQCITSYWNNTRCAAFVNDYSLVSYIVTYLGGDLPEECMAVYRDLVAHVEALAQLVDDFTLPGPELGGQAQAELNHLMRDPALLPPLVWDCDGLMRHAALDRHRDCRIDAGEHEPVYAAACNVATADFNRNDGRLLHNTQARAADAADDRPHRPADWTVHHKIYYYVLVPAFSRGRCCTAGVRFDRVYATLQNMVVPEIAPGEECPSDPVTDPAHPLHPANLVANTVNAMFHNGRVVVDGPAMLTLQVLAHNMAERTTALLCSAAPDAGANTASTANMRIFDGALHAGVLLMAPQHLDHTIQNGEYFYVLPVHALFAGADHVANAPNFPPALRDLARHVPLVPPALGANYFSSIRQPVVQHARESAAGENALTYALMAGYFKMSPVALYHQLKTGLHPGFGFTVVRQDRFVTENVLFSERASEAYFLGQLQVARHETGGGVSFTLTQPRGNVDLGVGYTAVAATATVRNPVTDMGNLPQNFYLGRGAPPLLDNAAAVYLRNAVVAGNRLGPAQPLPVFGCAQVPRRAGMDHGQDAVCEFIATPVATDINYFRRPCNPRGRAAGGVYAGDKEGDVIALMYDHGQSDPARPFAATANPWASQRFSYGDLLYNGAYHLNGASPVLSPCFKFFTAADITAKHRCLERLIVETGSAVSTATAASDVQFKRPPGCRELVEDPCGLFQEAYPITCASDPALLRSARDGEAHARETHFTQYLIYDASPLKGLSLSEQ ID NO: 22 = predicted sequence for ICP1/2 encoded by UL36MIPAALPHPTMKRQGDRDIVVTGVRNQFATDLEPGGSVSCMRSSLSFLSLLFDVGPRDVLSAEAIEGCLVEGGEWTRAAAGSGPPRMCSIIELPNFLEYPAARGGLRCVFSRVYGEVGFFGEPTAGLLETQCPAHTFFAGPWAMRPLSYTLLTIGPLGMGLYRDGDTAYLFDPHGLPAGTPAFIAKVRAGDVYPYLTYYAHDRPKVRWAGAMVFFVPSGPGAVAPADLTAAALHLYGASETYLQDEPFVERRVAITHPLRGEIGGLGALFVGVVPRGDGEGSGPVVPALPAPTHVQTPGADRPPEAPRGASGPPDTPQAGHPNRPPDDVWAAALEGTPPAKPSAPDAAASGPPHAAPPPQTPAGDAAEEAEDLRVLEVGAVPVGRHRARYSTGLPKRRRPTWTPPSSVEDLTSGERPAPKAPPAKAKKKSAPKKKAPVAAEVPASSPTPIAATVPPAPDTPPQSGQGGGDDGPASPSSPSVLETLGARRPPEPPGADLAQLFEVHPNVAATAVRLAARDAALAREVAACSQLTINALRSPYPAHPGLLELCVIFFFERVLAFLIENGARTHTQAGVAGPAAALLDFTLRMLPRKTAVGDFLASTRMSLADVAAHRPLIQHVLDENSQIGRLALAKLVLVARDVIRETDAFYGDLADLDLQLRAAPPANLYARLGEWLLERSRAHPNTLFAPATPTHPEPLLHRIQALAQFARGEEMRVEAEAREMREALDALARGVDSVSQRAGPLTVMPVPAAPGAGGRAPCPPALGPEAIQARLEDVRIQARRAIESAVKEYFHRGAVYSAKALQASDSHDCRFHVASAAVVPMVQLLESLPAFDQHTRDVAQRAALPPPPPLATSPQAILLRDLLQRGQPLDAPEDLAAWLSVLTDAATQGLIERKPLEELARSIHGINDQQARRSSGLAELQRFDALDAALAQQLDSDAAFVPATGPAPYVDGGGLSPEATRMAEDALRQARAMEAAKMTAELAPEARSRLRERAHALEAMLNDARERAKVAHDAREKFLHKLQGVLRPLPDFVGLKACPAVLATLRASLPAGWTDLADAVRGPPPEVTAALRADLWGLLGQYREALEHPTPDTATALAGLHPAFVVVLKTLFADAPETPVLVQFFSDHAPTIAKAVSNAINAGSAAVATASPAATVDAAVRAHGALADAVSALGAAARDPASPLSFLAVLADSAAGYVKATRLALEARGAIDELTTLGSAAADLVVQARRACAQPEGDHAALIDAAARATTAARESLAGHEAGFGGLLHAEGTAGDHSPSGRALQELGKVIGATRRRADELEAAVADLTAKMAAQRARGSSERWAAGVEAALDRVENRAEFDVVELRRLQALAGTHGYNPRDFRKRAEQALAANAEAVTLALDTAFAFNPYTPENQRHPMLPPLAAIHRLGWSAAFHAAAETYADMFRVDAEPLARLLRIAEGLLEMAQAGDGFIDYHEAVGRLADDMTSVPGLRRYVPFFQHGYADYVELRDRLDAIRADVHRALGGVPLDLAAAAEQISAARNDPEATAELVRTGVTLPCPSEDALVACAAALERVDQSPVKNTAYAEYVAFVTRQDTAETKDAVVRAKQQRAEATERVMAGLREALAARERRAQIEAEGLANLKTMLKVVAVPATVAKTLDQARSVAEIADQVEVLLDQTEKTRELDVPAVIWLEHAQRTFETHPLSAARGDGPGPLARHAGRLGALFDTRRRVDALRRSLEEAEAEWDEVWGRFGRVRGGAWKSPEGFRAMHEQLRALQDTTNTVSGLRAQPAYERLSARYQGVLGAKGAERAEAVEELGARVTKHTALCARLRDEVVRRVPWEMNFDALGGLLAEFDAAAADLAPWAVEEFRGARELIQYRMGLYSAYARAGGQTGAGAESAPAPLLVDLRALDARARASSSPEGHEVDPQLLRRRGEAYLRAGGDPGPLVLREAVSALDLPFATSFLAPDGTPLQYALCFPAVTDKLGALLMRPEAACVRPPLPTDVLESAPTVTAMYVLTVVNRLQLALSDAQAANFQLFGRFVRHRQATWGASMDAAAELYVALVATTLTREFGCRWAQLGWASGAAAPRPPPGPRGSQRHCVAFNENDVLVALVAGVPEHIYNFWRLDLVRQHEYMHLTLERAFEDAAESMLFVQRLTPHPDARIRVLPTFLDGGPPTRGLLFGTRLADWRRGKLSETDPLAPWRSALELGTQRRDVPALGKLSPAQALAAVSVLGRMCLPSAALAALWTCMFPDDYTEYDSFDALLAARLESGQTLGPAGGREASLPEAPHALYRPTGQHVAVLAAATHRTPAARVTAMDLVLAAVLLGAPVVVALRNTTAFSRESELELCLTLFDSRPGGPDAALRDVVSSDIETWAVGLLHTDLNPIENACLAAQLPRLSALIAERPLADGPPCLVLVDISMTPVAVLWEAPEPPGPPDVRFVGSEATEELPFVATAGDVLAASAADADPFFARAILGRPFDASLLTGELFPGHPVYQRPLADEAGPSAPTAARDPRDLAGGDGGSGPEDPAAPPARQADPGVLAPTLLTDATTGEPVPPRMWAWIHGLEELASDDAGGPTPNPAPALLPPPATDQSVPTSQYAPRPIGPAATARETRPSVPPQQNTGRVPVAPRDDPRPSPPTPSPPADAALPPPAFSGSAAAFSAAVPRVRRSRRTRAKSRAPRASAPPEGWRPPALPAPVAPVAASARPPDQPPTPESAPPAWVSALPLPPGPASARGAFPAPTLAPIPPPPAEGAVVPGGDRRRGRRQTTAGPSPTPPRGPAAGPPRRLTRPAVASLSASLNSLPSPRDPADHAAAVSAAAAAVPPSPGLAPPTSAVQTSPPPLAPGPVAPSEPLCGWVVPGGPVARRPPPQSPATKPAARTRIRARSVPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPPVTRTLTPQSRDSVPTPESPTHTNTHLPVSAVTSWASSLALHVDSAPPPASLLQTLHISSDDEHSDADSLRFSDSDDTEALDPLPPEPHLPPADEPPGPLAADHLQSPHSQFGPLPVQANAVLSRRYVRSTGRSALAVLIRACRRIQQQLQRTRRALFQRSNAVLTSLHHVRMLLG SEQ ID NO: 23 =ICP1/2 internal fragment encoded by construct UL36.3.4.1AAQRARGSSERWAAGVEAALDRVENRAEFDVVELRRLQALAGTHGYNPRDFRKRAEQALAANAEAVTLALDTAFAFNPYTPENQRHPMLPPLAAIHRLGWSAAFHAAAETYADMFRVDAEPLARLLRIAEGLLEMAQAGDGFIDYHEAVGRLADDMTSVPGLRRYVPFFQHGYADYVELRDRLDAIRADVHRALGGVPLDLAAAAEQISAARNDPEATAELVRTGVTLPCPSEDALVACAAALERVDQSPVKNTAYAEYVAFVTRQDTAETKDAVVRAKQQRAEATERVMAGLREALAARERRAQIEAEGLANLKTMLKVVAVPATVAKTLDQARSVAEIADQVEVLLDQTEKTRELDVPAVIWLEHAQRTFETHPLSAARGDGPGPLARHAGRLGALFDTRRRVDALRRSLEEAEAEWDEVWGRFGRVRGGAWKSPEGFRAMHEQLRALQDTTNTVSGLRAQPAYERLSARYQGVLGAKGAERAEAVEELGARVTKHTALCARLRDEVVRRVPWEMNFDALGGLLAEFDAAAADLAPWAVEEFRGARELIQYRMGLYSAYARAGGQTGAGAESAPAPLLVDLRALDARARASSSPEGHEVDPQLLRRRGEAYLRAGGDPGPLVLREAVSALDLPFATSFLAPDGTPLQYALCFPAVTDKLGALLMRPEAACVRPPLPTDVLESAPTVTAMYVLTVVNRLQLALSDAQAANFQLFGRFVRHRQATWGASMDAAAELYVALVATTLTREFGCRWAQLGWASGAAAPRPPPGPRGSQRHCVAFNENDVLVALVAGVPEHIYNFWRLDLVRQHEYMHLTLERAFEDAAESMLFVQRLTPHPDARIRVLPTFLDGGPPTRGLLFGTRLADWRRGKLSETDPLAPWRSALELGTQRRDVPALGKLSPAQALAAVSVLGRMCLPSAALAALWTCMFPDDYTEYDSFDALLAARLESGQTLGPAGGREASL SEQ ID NO: 24 =ICP1/2 internal fragment encoded by construct UL36.4.2.5EYDSFDALLAARLESGQTLGPAGGREASLPEAPHALYRPTGQHVAVLAAATHRTPAARVTAMDLVLAAVLLGAPVVVALRNTTAFSRESELELCLTLFDSRPGGPDAALRDVVSSDIETWAVGLLHTDLNPIENACLAAQLPRLSALIAERPLADGPPCLVLVDISMTPVAVLWEAPEPPGPPDVRFVGSEATEELPFVATAGDVLAASAADADPFFARAILGRPFDASLLTGELFPGHPVYQRPLADEAGPSAPTAARDPRDLAGGDGGSGPEDPAAPPARQADPGVLAPTLLTDATTGEPVPPRMWAWIHGLEELASDDAGGPTPNPAPALLPPPATDQSVPTSQYAPRPIGPAATARETRPSVPPQQNTGRVPVAPRDDPRPSPPTPSPPADAALPPPAFSGSAAAFSAAVPRVRRSRRTRAKSRAPRASAPPEGWRPPALPAPVAPVAASARPPDQPPTPESAPPAWVSALPLPPGPASARGAFPAPTLAPIPPPPAEGAVVPGGDRRRGRRQTTAGPSPTPPRGPAAGPPRRLTRPAVASLSASLNSLPSPRDPADHAAAVSAAAAAVPPSPGLAPPTSAVQTSPPPLAPGPVAPSEPLCGWVVPGGPVARRPPPQSPATKPAARTRIRARSVPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPQPPLPPVTRTLTPQSRDSVPTPESPTHTNTHLPVSAVTSWASSLALHVDSAPPPASLLQTLHISSDDEHSDADSLRFSDSDDTEALDPLPPEPHLPPADEPPGPLAADHLQSPHSQFGPLPVQANAVLSRRYVRSTGRSALAVLIRACRRIQQQLQRTRRALFQRSNAVLTSLHHVRMLLG SEQ ID NO: 25 =predicted sequence for reductase encoded by UL40MDPAVSPASTDPLDTHASGAGAAPIPVCPTPERYFYTSQCPDINHLRSLSILNRWLETELVFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKDILHYYVEQECIEVVHSRVYNIIQLVLFHNNDQARRAYVARTINHPAIRVKVDWLEARVRECDSIPEKFILMILIEGVFFAASFAAIAYLRTNNLLRVTCQSNDLISRDEAVHTTASCYIYNNYLGGHAKPEAARVYRLFREAVDIEIGFIRSQAPTDSSILSPGALAAIENYVRFSADRLLGLIHMQPLYSAPAPDASFPLSLMSTDKHTNFFECRSTSYAGAVVNDL SEQ ID NO: 26 = ICP47 encoded by US12MSWALKTTDMFLDSSRCTHRTYGDVCAEIHKREREDREAARTAVTDPELPLLCPPDVRSDPASRNPTQQTRGCARSNERQDRVLAP SEQ ID NO: 27 = gM2 encoded by UL10MGRRAPRGSPEAAPGADVAPGARAAWWVWCVQVATFIVSAICVVGLLVLASVFRDRFPCLYAPATSYAKANATVEVRGGVAVPLRLDTQSLLATYAITSTLLLAAAVYAAVGAVTSRYERALDAARRLAAARMAMPHATLIAGNVCAWLLQITVLLLAHRISQLAHLIYVLHFACLVYLAAHFCTRGVLSGTYLRQVHGLIDPAPTHHRIVGPVRAVMTNALLLGTLLCTAAAAVSLNTIAALNFNFSAPSMLICLTTLFALLVVSLLLVVEGVLCHYVRVLVGPHLGAIAATGIVGLACEHYHTGGYYVVEQQWPGAQTGVRVALALVAAFALAMAVLRCTRAYLYHRRHHTKFFVRMRDTRHRAHSALRRVRSSMRGSRRGGPPGDPGYAETPYASVSHHAEIDRYGDSDGDPIYDEVAPDHEAELYARVQRPGPVPDAEPIYDTVEGYAPRSAGEPVYSTVRRW SEQ ID NO: 28 =predicted sequence for cleavage/packaging protein encoded by UL15MFGQQLASDVQQYLERLEKQRQQKVGVDEASAGLTLGGDALRVPFLDFATATPKRHQTVVPGVGTLHDCCEHSPLFSAVARRLLFNSLVPAQLRGRDFGGDHTAKLEFLAPELVRAVARLRFRECAPEDAVPQRNAYYSVLNTFQALHRSEAFRQLVHFVRDFAQLLKTSFRASSLAETTGPPKKRAKVDVATHGQTYGTLELFQKMILMHATYFLAAVLLGDHAEQVNTFLRLVFEIPLFSDTAVRHFRQRATVFLVPRRHGKTWFLVPLIALSLASFRGIKIGYTAHIRKATEPVFDEIDACLRGWFGSSRVDHVKGETISFSFPDGSRSTIVFASSHNTNGIRGQDFNLLFVDEANFIRPDAVQTIMGFLNQANCKIIFVSSTNTGKASTSFLYNLRGAADELLNVVTYICDDHMPRVVTHTNATACSCYILNKPVFITMDGAVRRTADLFLPDSFMQEIIGGQARETGDDRPVLTKSAGERFLLYRPSTTTNSGLMAPELYVYVDPAFTANTRASGTGIAVVGRYRDDFIIFALEHFFLRALTGSAPADIARCVVHSLAQVLALHPGAFRSVRVAVEGNSSQDSAVAIATHVHTEMHRILASAGANGPGPELLFYHCEPPGGAVLYPFFLLNKQKTPAFEYFIKKFNSGGVMASQELVSVTVRLQTDPVEYLSEQLNNLIETVSPNTDVRMYSGKRNGAADDLMVAVIMAIYLAAPTGIPPAFFPITRTS SEQ ID NO: 29 =predicted sequence for ICP35 encoded by UL26.5MNPVSASGAPAPPPPGDGSYLWIPASHYNQLVTGQSAPRHPPLTACGLPAAGTVAYGHPGAGPSPHYPPPPAHPYPGMLFAGPSPLEAQIAALVGAIAADRQAGGLPAAAGDHGIRGSAKRRRHEVEQPEYDCGRDEPDRDFPYYPGEARPEPRPVDSRRAARQASGPHETITALVGAVTSLQQELAHMRARTHAPYGPYPPVGPYHHPHADTETPAQPPRYPAKAVYLPPPHIAPPGPPLSGAVPPPSYPPVAVTPGPAPPLHQPSPAHAHPPPPPPGPTPPPAASLPQPEAPGAEAGALVNASSAAHVNVDTARAADLFVSQMMGSR SEQ ID NO: 30 =predicted sequence for polymerase encoded by UL30MFCAAGGPASPGGKPAARAASGFFAPHNPRGATQTAPPPCRRQNFYNPHLAQTGTQPKALGPAQRHTYYSECDEFRFIAPRSLDEDAPAEQRTGVHDGRLRRAPKVYCGGDERDVLRVGPEGFWPRRLRLWGGADHAPEGFDPTVTVFHVYDILEHVEHAYSMRAAQLHERFMDAITPAGTVITLLGLTPEGHRVAVHVYGTRQYFYMNKAEVDRHLQCRAPRDLCERLAAALRESPGASFRGISADHFEAEVVERADVYYYETRPTLYYRVFVRSGRALAYLCDNFCPAIRKYEGGVDATTRFILDNPGFVTFGWYRLKPGRGNAPAQPRPPTAFGTSSDVEFNCTADNLAVEGAMCDLPAYKLMCFDIECKAGGEDELAFPVAERPEDLVIQISCLLYDLSTTALEHILLFSLGSCDLPESHLSDLASRGLPAPVVLEFDSEFEMLLAFMTFVKQYGPEFVTGYNIINFDWPFVLTKLTEIYKVPLDGYGRMNGRGVFRVWDIGQSHFQKRSKIKVNGMVNIDMYGIITDKVKLSSYKLNAVAEAVLKDKKKDLSYRDIPAYYASGPAQRGVIGEYCVQDSLLVGQLFFKFLPHLELSAVARLAGINITRTIYDGQQIRVFTCLLRLAGQKGFILPDTQGRFRGLDKEAPKRPAVPRGEGERPGDGNGDEDKDDDEDGDEDGDEREEVARETGGRHVGYQGARVLDPTSGFHVDPVVVFDFASLYPSIIQAHNLCFSTLSLRPEAVAHLEADRDYLEIEVGGRRLFFVKAHVRESLLSILLRDWLAMRKQIRSRIPQSTPEEAVLLDKQQAAIKVVCNSVYGFTGVQHGLLPCLHVAATVTTIGREMLLATRAYVHARWAEFDQLLADFPEAAGMRAPGPYSMRIIYGDTDSIFVLCRGLTAAGLVAMGDKMASHISRALFLPPIKLECEKTFTKLLLIAKKKYIGVICGGKMLIKGVDLVRKNNCAFINRTSRALVDLLFYDDTVSGAAAALAERPAEEWLARPLPEGLQAFGAVLVDAHRRITDPERDIQDFVLTAELSRHPRAYTNKRLAHLTVYYKLMARRAQVPSIKDRIPYVIVAQTREVEETVARLAALRELDAAAPGDEPAPPAALPSPAKRPRETPSHADPPGGASKPRKLLVSELAEDPGYAIARGVPLNTDYYFSHLLGAACVTFKALFGNNAKITESLLKRFIPETWHPPDDVAARLRAAGFGPAGAGATAEETRRMLHRAFDTLA SEQ ID NO: 31 =predicted sequence for helicase/primase complex encoded by UL5MAASGGEGSRDVRAPGPPPQQPGARPAVRFRDEAFLNFTSMHGVQPIIARIRELSQQQLDVTQVPRLQWFRDVAALEVPTGLPLREFPFAAYLITGNAGSGKSTCVQTLNEVLDCVVTGATRIAAQNMYVKLSGAFLSRPINTIFHEFGFRGNHVQAQLGQHPYTLASSPASLEDLQRRDLTYYWEVILDITKRALAAHGGEDARNEFHALTALEQTLGLGQGALTRLASVTHGALPAFTRSNIIVIDEAGLLGRHLLTTVVYCWWMINALYHTPQYAGRLRPVLVCVGSPTQTASLESTFEHQKLRCSVRQSENVLTYLICNRTLREYTRLSHSWAIFINNKRCVEHEFGNLMKVLEYGLPITEEHMQFVDRFVVPESYITNPANLPGWTRLFSSHKEVSAYMAKLHAYLKVTREGEFVVFTLPVLTFVSVKEFDEYRRLTQQPTLTMEKWITANASRITNYSQSQDQDAGHVRCEVHSKQQLVVARNDITYVLNSQVAVTARLRKMVFGFDGTFRTFEAVLRDDSFVKTQGETSVEFAYRFLSRLMFGGLIHFYNFLQRPGLDATQRTLAYGRLGELTAELLSLRRDAAGASATRAADTSDRSPGERAFNFKHLGPRDGGPDDFPDDDLDVIFAGLDEQQLDVFYCHYALEEPETTAAVHAQFGLLKRAFLGRYLILRELFGEVFESAPFSTYVDNVIFRGCELLTGSPRGGLMSVALQTDNYTLMGYTYTRVFAFAEELRRRHATAGVAEFLEESPLPYIVLRDQHGFMSVVNTNISEFVESIDSTELAMAINADYGISSKLAMTITRSQGLSLDKVAICFTPGNLRLNSAYVAMSRTTSSEFLHMNLNPLRERHERDDVISEHILSALRDPNVVIVY SEQ ID NO: 32 =predicted sequence for helicase/primase complex encoded by UL8MEAPGIVWVEESVSAITLYAVWLPPRTRDCLHALLYLVCRDAAGEARARFAEVSVGSSDLQDFYGSPDVSAPGAVAAARAATAPAASPLEPLGDPTLWRALYACVLAALERQTGRWALFVPLRLGWDPQTGLVVRVERASWGPPAAPRAALLDVEAKVDVDPLALSARVAEHPGARLAWARLAAIRDSPQCASSASLAVTITTRTARFAREYTTLAFPPTRKEGAFADLVEVCEVGLRPRGHPQRVTARVLLPRGYDYFVSAGDGFSAPALVALFRQWHTTVHAAPGALAPVFAFLGPGFEVRGGPVQYFAVLGFPGWPTFTVPAAAAAESARDLVRGAAATHAACLGAWPAVGARVVLPPRAWPAVASEAAGRLLPAFREAVARWHPTATTIQLLDPPAAVGPVWTARFCFSGLQAQLLAALAGLGEAGLPEARGRAGLERLDALVAAAPSEPWARAVLERLVPDACDACPALRQLLGGVMAAVCLQIEQTASSVKFAVCGGTGAAFWGLFNVDPGDADAAHGAIQDARRALEASVRAVLSANGIRPRLAPSLAPEGVYTHVVTWSQTGAWFWNSRDDTDFLQGFPLRGAAYAAAAEVMRDALRRILRRPAAGPPEEAVCAARGVMEDACDRFVLDAFGRRLDAEYWSVLTPPGEADDPLPQTAFRGGALLDAEQYWRRVVRVCPGGGESVGVPVDLYPRPLVLPPVDCAHHLREILREIQLVFTGVLEGVWGEGGSFVYPFDEKIRFLFPSEQ ID NO: 33 = predicted sequence for unknown protein encoded by UL15.5MDGAVRRTADLFLPDSFMQEIIGGQARETGDDRPVLTKSAGERFLLYRPSTTTNSGLMAPELYVYVDPAFTANTRASGTGIAVVGRYRDDFIIFALEHFFLRALTGSAPADIARCVVHSLAQVLALHPGAFRSVRVAVEGNSSQDSAVAIATHVHTEMHRILASAGANGPGPELLFYHCEPPGGAVLYPFFLLNKQKTPAFEYFIKKFNSGGVMASQELVSVTVRLQTDPVEYLSEQLNNLIETVSPNTDVRMYSGKRNGAADDLMVAVIMAIYLAAPTGIPPAFFPITRTSSEQ ID NO: 34 = predicted sequence for packaging protein encoded by UL32MATSAPGVPSSAAVREESPGSSWKEGAFERPYVAFDPDLLALNEALCAELLAACHVVGVPPASALDEDVESDVAPAPPRPRGAAREASGGRGPGSARGPPADPTAEGLLDTGPFAAASVDTFALDRPCLVCRTIELYKQAYRLSPQWVADYAFLCAKCLGAPHCAASIFVAAFEFVYVMDHHFLRTKKATLVGSFARFALTINDIHRHFFLHCCFRTDGGVPGRHAQKQPRPTPSPGAAKVQYSNYSFLAQSATRALIGTLASGGDDGAGAGAGGGSGTQPSLTTALMNWKDCARLLDCTEGKRGGGDSCCTRAAARNGEFEAAAGALAQGGEPETWAYADLILLLLAGTPAVWESGPRLRAAADARRAAVSESWEAHRGARMRDAAPRFAQFAEPQPQPDLDLGPLMATVLKHGRGRGRTGGECLLCNLLLVRAYWLAMRRLRASVVRYSENNTSLFDCIVPVVDQLEADPEAQPGDGGRFVSLLRAAGPEAIFKHMFCDPMCAITEMEVDPWVLFGHPRADHRDELQLHKAKLACGNEFEGRVCIALRALIYTFKTYQVFVPKPTALATFVREAGALLRRHSISLLSLEHTLCTYVSEQ ID NO: 35 =predicted sequence for ICP1/2 fragment encoded by construct UL36.4.2MEYDSFDALLAARLESGQTLGPAGGREASLPEAPHALYRPTGQHVAVLAAATHRTPAARVTAMDLVLAAVLLGAPVVVALRNTTAFSRESELELCLTLFDSRPGGPDAALRDVVSSDIETWAVGLLHTDLNPIENACLAAQLPRLSALIAERPLADGPPCLVLVDISMTPVAVLWEAPEPPGPPDVRFVGSEATEELPFVATAGDVLAASAADADPFFARAILGRPFDASLLTGELFPGHPVYQRPLADEAGPSAPTAARDPRDLAGGDGGSGPEDPAAPPARQADPGVLAPTLLTDATTGEPVPPRMWANIHGLEELASDDAGGPT SEQ ID NO: 36 =predicted sequence for ICP27 encoded by UL54MATDIDMLIDLGLDLSDSELEEDALERDEEGRRDDPESDSSGECSSSDEDMEDPCGDGGAEAIDAAIPKGPPARPEDAGTPEASTPRPAARRGADDPPPATTGVWSRLGTRRSASPREPHGGKVARIQPPSTKAPHPRGGRRGRRRGRGRYGPGGADSTPKPRRRVSRNAHNQGGRHPASARTDGPGATHGEARRGGEQLDVSGGPRPRGTRQAPPPLMALSLTPPHADGRAPVPERKAPSADTIDPAVRAVLRSISERAAVERISESFGRSALVMQDPFGGMPFPAANSPWAPVLATQAGGFDAETRRVSWETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLF SEQ ID NO: 37 =virion protein encoded by UL49.5MTGKPARLGRWVVLLFVALVAGVPGEPPNAAGARGVIGDAQCRGDSAGVVSVPGVLVPFYLGMTSMGVCMIAHVYQICQRALAAGSA SEQ ID NO: 38 = gG2 encoded by US4NRWGSGVPGPINPPNSDVVFPGGSPVAQYCYAYPRLDDPGPLGSADAGRQDLPRRVVRHEPLGRSFLTGGLVLLAPPVRGFGAPNATYAARVTYYRLTRACRQPILLRQYGGCRGGEPPSPKTCGSYTYTYQGGGPPTRYALVNASLLVPIWDRAAETFEYQIELGGELHVGLLWVEVGGEGPGPTAPPQAARAEGGPCVPPVPAGRPWRSVPPVWYSAPNPGFRGLRFRERCLPPQTPAAPSDLPRVAFAPQSLLVGITGRTFIRMARPTEDVGVLPPHWAPGALDDGPYAPFPPRPRFRRSEQ ID NO: 39 = RS1ATGTCGTACTACCATCACCATCACCATCACAGTGCCGAACAGCGTAAAAAGAAAAAAACCACCACCACGACCCAAGGACGTGGAGCTGAAGTTGCTATGGCGGATGAGGATGGAGGCCGCTTGAGAGCTGCTGCTGAGACTACTGGAGGACCTGGATCACCGGACCCTGCCGATGGACCCCCCCCTACACCAAACCCCGATCGTAGACCGGCTGCTAGACCTGGATTCGGATGGCATGGAGGACCCGAGGAAAACGAGGACGAGGCGGACGACGCCGCTGCCGACGCCGACGCCGATGAGGCTGCCCCTGCTTCTGGAGAGGCGGTAGACGAACCTGCTGCCGATGGAGTTGTTAGCCCTAGGCAATTGGCTTTGTTGGCGAGCATGGTAGACGAGGCTGTGAGAACAATCCCTTCCCCTCCCCCTGAACGTGATGGAGCACAAGAGGAGGCGGCTAGGAGTCCCTCACCACCCCGTACACCTTCTATGAGAGCGGATTACGGCGAGGAAAACGACGACGACGACGATGATGATGACGACGATGATCGTGATGCCGGACGCTGGGTTAGGGGACCTGAAACCACTTCTGCTGTCCGTGGAGCATACCCCGATCCTATGGCGAGTTTGAGCCCTAGACCACCTGCCCCGAGGAGACACCACCACCACCACCATCATAGGCGTAGACGTGCTCCTAGACGTCGTTCTGCCGCTAGTGACTCTTCCAAATCTGGCTCTTCTTCATCTGCCTCTTCCGCTTCATCTTCGGCCTCATCGTCCTCTTCGGCATCCGCTTCGAGTAGTGATGATGATGATGACGACGACGCTGCTAGAGCCCCCGCTTCTGCTGCCGACCACGCTGCTGGCGGAACTTTGGGAGCCGACGACGAGGAGGCGGGAGTTCCTGCTCGTGCCCCGGGAGCTGCTCCGAGGCCTTCTCCACCCCGTGCTGAACCTGCTCCGGCTAGAACACCGGCCGCTACTGCTGGTAGACTGGAGCGTAGACGTGCCCGTGCTGCTGTGGCTGGTAGAGATGCTACTGGCCGCTTCACTGCTGGCCGTCCTAGACGTGTTGAACTGGACGCCGATGCTGCTTCTGGTGCTTTCTACGCCCGTTACCGTGATGGTTACGTGTCTGGTGAACCTTGGCCTGGCGCTGGTCCACCTCCGCCCGGACGTGTACTCTACGGTGGATTGGGCGATTCTCGCCCTGGTCTGTGGGGCGCTCCGGAGGCTGAGGAGGCTAGAGCCCGTTTCGAGGCTTCTGGTGCCCCTGCTCCTGTTTGGGCTCCTGAATTGGGCGACGCTGCTCAACAATACGCCCTCATCACACGCTTGCTGTACACTCCCGACGCCGAGGCTATGGGATGGCTCCAAAACCCTAGAGTTGCCCCTGGTGATGTTGCTCTGGATCAGGCTTGTTTCCGTATCTCCGGCGCTGCTCGTAACTCTTCTTCGTTCATCTCCGGTTCTGTGGCTAGAGCTGTGCCTCACTTGGGATACGCCATGGCCGCTGGACGTTTCGGCTGGGGACTGGCTCATGTTGCTGCCGCTGTAGCAATGTCTAGACGCTACGACCGTGCTCAAAAAGGATTCTTGCTCACGTCACTGAGGCGTGCTTACGCCCCTTTGTTGGCCCGTGAAAACGCTGCCCTCACTGGCGCCCGTACCCCCGATGACGGTGGCGACGCCAACCGCCACGATGGTGATGATGCTAGAGGCAAACCCGCTGCCGCTGCTGCTCCTTTGCCCTCTGCCGCCGCTTCCCCTGCCGATGAACGTGCTGTTCCTGCCGGTTACGGTGCCGCTGGTGTGTTGGCTGCTTTGGGACGCTTGAGTGCTGCCCCGGCTAGTGCCCCCGCTGGTGCCGATGACGATGACGATGACGATGGTGCTGGCGGAGGCGGTGGCGGTAGACGTGCTGAGGCTGGACGTGTTGCTGTTGAATGCCTGGCTGCCTGTAGAGGAATCTTGGAGGCTCTGGCCGAGGGATTCGACGGAGACTTGGCGGCTGTACCGGGACTGGCGGGAGCGAGGCCTGCCGCTCCACCTCGCCCCGGTCCTGCTGGTGCTGCCGCTCCTCCTCATGCCGACGCTCCTAGACTCCGTGCTTGGCTCCGTGAACTCCGTTTCGTTCGTGACGCTTTGGTTCTGATGAGACTGAGAGGCGACTTGAGAGTGGCTGGAGGATCCGAGGCTGCTGTTGCTGCTGTCCGTGCTGTTTCTTTGGTTGCTGGTGCTTTGGGCCCTGCTTTGCCGAGATCTCCCCGTTTGTTGTCGAGTGCCGCCGCTGCTGCCGCCGATTTGTTGTTCCAAAACCAATCCCTCCGCCCTCTGCTCGCCGACACTGTTGCCGCTGCCGATTCTCTGGCTGCTCCGGCTTCTGCCCCACGTGAAGCTCGTAAACGTAAATCACCCGCTCCGGCTCGTGCTCCCCCTGGTGGCGCCCCTAGACCCCCTAAAAAATCCCGTGCCGATGCCCCTAGACCTGCTGCTGCTCCCCCCGCTGGTGCTGCTCCCCCCGCTCCCCCTACTCCCCCCCCACGCCCACCTCGTCCCGCTGCCCTCACACGCCGTCCTGCTGAGGGACCCGATCCACAAGGCGGCTGGCGTAGACAACCTCCTGGCCCATCCCATACACCGGCACCATCTGCCGCTGCTTTGGAGGCTTACTGTGCTCCTCGTGCTGTGGCTGAACTCACCGATCATCCGCTGTTCCCTGCTCCCTGGCGTCCCGCCCTCATGTTCGATCCTAGAGCTTTGGCTTCCTTGGCCGCTCGTTGTGCTGCCCCTCCCCCTGGCGGTGCTCCGGCTGCTTTCGGTCCTCTCCGTGCCTCTGGTCCACTCCGCCGTGCCGCTGCCTGGATGAGACAAGTTCCCGACCCTGAGGATGTTAGAGTTGTGATCTTGTACTCGCCCTTGCCTGGCGAGGATTTGGCCGCTGGTAGAGCTGGCGGTGGCCCCCCTCCTGAATGGTCTGCTGAACGTGGTGGTTTGTCTTGCTTGTTGGCCGCCCTGGGAAACCGTCTGTGTGGTCCTGCTACTGCTGCTTGGGCTGGAAACTGGACTGGCGCTCCCGATGTTTCTGCTCTCGGTGCTCAAGGAGTTTTGCTGCTCTCTACTCGTGACTTGGCATTCGCTGGAGCTGTTGAATTCCTGGGACTCTTGGCTGGCGCTTGTGATAGGAGACTCATCGTCGTAAACGCTGTGAGAGCTGCCGATTGGCCTGCCGATGGTCCTGTTGTGTCTCGTCAACACGCTTACTTGGCTTGTGAAGTGTTGCCCGCTGTCCAATGTGCTGTTCGCTGGCCTGCTGCTCGTGATCTGAGGCGTACTGTTCTGGCTAGTGGTCGTGTTTTCGGACCTGGTGTTTTCGCTCGTGTCGAAGCTGCTCACGCTAGACTGTACCCCGATGCCCCACCCCTCCGTTTGTGTCGTGGAGCAAACGTTCGCTACCGTGTCCGTACTCGTTTCGGACCCGATACTCTGGTTCCAATGTCCCCTCGTGAATACCGTCGTGCTGTTCTGCCTGCCCTCGATGGACGTGCTGCCGCTTCTGGCGCTGGTGACGCTATGGCTCCTGGCGCTCCGGACTTCTGTGAGGATGAGGCTCACTCACATCGTGCCTGTGCCCGCTGGGGACTGGGCGCTCCATTGAGGCCTGTATACGTGGCACTGGGCCGTGATGCTGTTAGAGGCGGACCCGCTGAATTGAGAGGCCCTCGTCGTGAATTCTGTGCTAGGGCTCTGCTCGAACCCGATGGAGATGCTCCTCCTTTGGTACTCCGTGACGACGCCGATGCTGGTCCTCCCCCACAAATTCGCTGGGCTAGTGCTGCTGGACGTGCTGGTACTGTATTGGCTGCTGCTGGCGGTGGCGTTGAAGTTGTTGGTACTGCCGCTGGACTCGCTACACCTCCCCGCCGTGAACCTGTAGACATGGATGCTGAACTCGAGGATGATGACGACGGATTGTTCGGAGAGTAATAGSEQ ID NO: 40 = construct US6ΔTMRATGAAGTTCCTCGTGAACGTGGCCCTGGTGTTCATGGTGGTGTACATCAGCTACATCTACGCCAACCGTTGGAAGTACGCTCTGGCTGACCCATCCCTGAAGATGGCTGACCCCAACCGTTTCCGTGGCAAGAACCTGCCCGTGCTGGACCAGCTGACCGACCCCCCTGGCGTGAAGCGTGTGTACCACATCCAGCCATCCCTCGAAGACCCCTTCCAGCCCCCCTCCATCCCCATCACCGTGTACTACGCTGTGCTGGAACGCGCTTGCCGTTCCGTGCTGCTGCACGCTCCTTCCGAGGCTCCCCAGATCGTGCGTGGTGCTTCCGACGAGGCTCGCAAGCACACCTACAACCTGACTATCGCTTGGTACAGGATGGGTGACAACTGCGCTATCCCTATCACCGTCATGGAATACACCGAGTGCCCCTACAACAAGTCCCTGGGCGTGTGCCCTATCCGTACCCAGCCCCGTTGGTCCTACTACGACTCCTTCAGCGCTGTGTCCGAGGACAACCTGGGTTTCCTGATGCACGCTCCCGCTTTCGAGACTGCTGGCACCTACCTGCGTCTGGTCAAGATCAACGACTGGACCGAGATCACCCAGTTCATCCTGGAACACCGTGCTCGTGCTTCGTGCAAGTACGCCCTGCCCCTGCGTATCCCTCCTGCTGCTTGCCTGACCTCCAAGGCTTACCAGCAGGGCGTGACCGTGGACTCCATCGGCATGCTGCCCCGTTTCATCCCCGAGAACCAGCGTACCGTGGCTCTGTACTCTCTGAAGATCGCTGGCTGGCACGGTCCTAAGCCCCCCTACACCTCCACTCTGCTGCCCCCTGAGCTGTCCGACACCACCAACGCTACTCAGCCCGAGTTGGTGCCTGAGGACCCCGAGGACTCCGCTCTGTTGGAGGACCCCGCTGGAACCGTGTCCTCCCAGATCCCCCCCAACTGGCACATCCCTTCCATCCAGGACGTGGCCCCTCACCACGCTCCAGCTGCTCCCTCCAACCCCCGTCGTCGTGCTCAGATGGCTCCCAAGCGTCTGCGTCTGCCCCACATCCGTGACGACGACGCTCCTCCATCCCACCAGCCCCTGTTCTACCACCACCACCATCACCACTAATAA SEQ ID NO: 41 = RL1ATGTCTCGTCGTCGTGGTCCTCGTCGTCGTGGTCCTCGTCGTCGTCCGCGTCCGGGTGCGCCGGCGGTACCACGCCCGGGTGCGCCGGCAGTGCCGCGTCCAGGCGCACTGCCTACCGCGGACTCTCAAATGGTGCCGGCGTATGATTCTGGTACTGCCGTCGAATCTGCTCCGGCAGCGAGCTCCCTGCTGCGTCGTTGGCTGCTGGTCCCTCAGGCGGACGATTCCGATGACGCAGACTACGCGGGCAACGACGACGCGGAGTGGGCTAACAGCCCGCCAAGCGAGGGTGGTGGCAAAGCGCCGGAGGCTCCGCACGCAGCGCCTGCCGCAGCGTGCCCGCCTCCGCCTCCTCGTAAAGAACGTGGCCCTCAACGTCCTCTGCCGCCGCACCTGGCTCTGCGTCTGCGTACTACCACTGAGTACCTGGCGCGTCTGTCTCTGCGTCGTCGCCGTCCGCCGGCTAGCCCGCCGGCCGATGCACCGCGTGGCAAAGTGTGCTTCTCTCCACGTGTTCAAGTTCGTCACCTGGTGGCTTGGGAAACGGCTGCCCGTCTGGCTCGCCGTGGCAGCTGGGCACGTGAGCGCGCAGACCGTGACCGCTTCCGTCGCCGTGTGGCGGCTGCTGAAGCCGTTATCGGCCCGTGCCTGGAACCTGAGGCTCGCGCTCGCGCGCGTGCGCGCGCTCGTGCCCACGAAGATGGCGGTCCAGCAGAGGAAGAAGAGGCAGCTGCAGCAGCGCGCGGTAGCTCCGCGGCTGCGGGTCCAGGTCGTCGTGCCGTA SEQ ID NO: 42 = RL2ATGTCGTACTACCATCACCATCACCATCACATGGAGCCACGTCCTGGTACTTCTTCTCGCGCTGATCCTGGTCCTGAACGTCCGCCACGCCAGACTCCGGGCACCCAGCCGGCCGCCCCTCACGCTTGGGGCATGCTGAACGATATGCAGTGGCTGGCGTCCTCTGATTCCGAAGAGGAGACTGAGGTTGGTATCAGCGATGATGATCTGCACCGCGACTCTACCAGCGAAGCAGGTTCCACTGACACCGAAATGTTTGAAGCGGGCCTGATGGATGCCGCGACCCCGCCGGCTCGTCCGCCGGCTGAACGTCAGGGTAGCCCTACGCCTGCGGATGCGCAAGGCTCTTGTGGTGGTGGTCCAGTAGGCGAAGAGGAGGCTGAGGCCGGTGGCGGCGGTGATGTGTGTGCGGTTTGTACCGATGAAATCGCACCGCCGCTGCGTTGTCAGTCTTTCCCGTGCCTGCACCCGTTTTGCATTCCGTGCATGAAAACCTGGATCCCGCTGCGCAACACTTGCCCGCTGTGCAACACTCCGGTTGCTTATCTGATCGTTGGTGTAACCGCATCTGGTTCCTTTTCTACCATCCCGATTGTCAACGACCCACGTACGCGTGTTGAGGCGGAGGCGGCTGTACGTGCGGGCACCGCGGTGGACTTTATCTGGACCGGTAACCCGCGCACCGCGCCACGCTCCCTGTCTCTGGGTGGCCATACCGTTCGTGCTCTGAGCCCGACCCCACCTTGGCCAGGCACCGATGACGAAGACGACGATCTGGCTGACGTTGACTATGTTCCGCCGGCACCGCGTCGCGCACCACGCCGTGGTGGCGGTGGCGCCGGTGCGACGCGCGGTACCTCCCAGCCGGCAGCAACTCGCCCAGCACCGCCGGGTGCCCCGCGTTCTAGCAGCTCCGGTGGCGCACCGCTGCGTGCTGGCGTGGGTTCTGGTTCCGGTGGTGGTCCGGCCGTGGCGGCTGTCGTCCCGCGTGTGGCTTCTCTGCCACCGGCAGCTGGTGGCGGTCGTGCTCAAGCTCGTCGTGTCGGCGAGGACGCAGCGGCTGCTGAGGGCCGTACTCCACCGGCCCGTCAACCGCGCGCAGCACAGGAACCGCCGATCGTGATCTCCGATTCCCCGCCACCGAGCCCGCGTCGCCCGGCGGGTCCGGGTCCGCTGTCTTTTGTATCCTCCAGCTCTGCTCAGGTAAGCAGCGGTCCTGGCGGTGGCGGCCTGCCACAGTCCTCTGGTCGTGCTGCTCGTCCTCGTGCGGCGGTTGCTCCTCGTGTACGTTCTCCGCCACGCGCTGCTGCCGCGCCGGTCGTTTCTGCCTCTGCTGACGCGGCAGGTCCGGCTCCGCCTGCAGTTCCGGTTGATGCACACCGTGCACCGCGCTCTCGTATGACCCAGGCGCAGACTGATACCCAGGCACAATCCCTGGGTCGCGCGGGTGCGACTGACGCTCGTGGTAGCGGTGGTCCGGGCGCTGAAGGTGGCCCGGGTGTTCCACGCGGTACTAACACTCCGGGCGCTGCGCCACACGCGGCTGAAGGTGCGGCTGCACGTCCGCGTAAACGTCGTGGTTCCGACAGCGGTCCGGCTGCAAGCAGCAGCGCGAGCTCTTCCGCTGCGCCTCGCAGCCCGCTGGCGCCGCAGGGTGTTGGCGCCAAGCGTGCTGCTCCGCGTCGTGCACCGGACTCCGATTCTGGCGACCGCGGTCACGGCCCGCTGGCCCCTGCTAGCGCAGGCGCTGCGCCGCCATCCGCCAGCCCGTCTTCTCAGGCAGCTGTGGCTGCGGCGTCCTCTTCTTCCGCTAGCAGCTCTTCCGCCTCTTCTAGCAGCGCGTCCTCTAGCAGCGCATCTTCCTCTTCTGCTTCTTCTTCTAGCGCTTCTAGCTCTTCCGCGTCCTCTTCCGCTGGCGGTGCAGGCGGCTCTGTTGCTTCCGCCAGCGGCGCAGGTGAGCGTCGTGAAACGAGCCTGGGCCCACGTGCTGCTGCACCGCGTGGCCCGCGTAAGTGTGCGCGCAAGACCCGCCACGCTGAAGGCGGTCCGGAGCCGGGTGCGCGTGATCCGGCTCCGGGTCTGACCCGTTACCTGCCGATTGCGGGTGTGTCCTCCGTTGTGGCACTGGCGCCGTATGTGAACAAAACTGTCACGGGCGATTGCCTGCCTGTTCTGGACATGGAAACCGGTCATATCGGCGCTTACGTCGTTCTGGTTGACCAAACCGGCAACGTGGCGGATCTGCTGCGTGCGGCCGCTCCGGCTTGGTCCCGTCGTACCCTGCTGCCGGAACATGCTCGCAACTGTGTACGCCCACCGGATTACCCAACCCCGCCGGCCTCCGAGTGGAACTCCCTGTGGATGACCCCGGTTGGTAACATGCTGTTCGACCAGGGCACGCTGGTTGGTGCTCTGGACTTTCACGGCCTGCGCTCCCGTCACCCGTGGTCCCGTGAGCAAGGCGCTCCGGCCCCTGCGGGCGATGCCCCGGCTGGCCACGGCGAGAGTACTAGAGGATCATAASEQ ID NO: 43 = construct UL36.3.4.1ATGTCGTACTACCATCACCATCACCATCACGCCGCTCAACGTGCTAGGGGATCCTCTGAACGCTGGGCTGCTGGTGTCGAGGCTGCTTTGGATAGAGTGGAGAACCGTGCCGAATTCGATGTTGTCGAGCTGAGGAGACTCCAAGCTTTGGCTGGTACTCACGGCTACAACCCTCGTGATTTCCGTAAACGTGCCGAACAGGCTTTGGCGGCAAACGCTGAGGCCGTAACATTGGCTCTGGACACTGCCTTCGCTTTCAACCCATACACGCCCGAAAACCAACGTCATCCTATGCTCCCACCTCTCGCTGCTATTCACCGCCTGGGATGGAGCGCTGCTTTCCATGCTGCTGCTGAAACTTACGCCGACATGTTCCGTGTCGATGCCGAACCACTGGCTAGACTGCTCCGTATCGCTGAGGGACTGCTGGAGATGGCTCAAGCTGGCGACGGATTCATCGATTACCATGAGGCTGTCGGTAGACTGGCCGATGATATGACTTCTGTGCCCGGATTGAGGCGCTACGTTCCTTTCTTCCAACATGGCTACGCCGATTACGTGGAACTGAGAGATCGCCTGGATGCTATTAGGGCCGACGTCCATAGAGCACTCGGTGGTGTTCCGCTGGATTTGGCGGCTGCTGCCGAACAAATTTCCGCTGCTCGTAACGATCCTGAGGCTACTGCTGAATTGGTCCGTACTGGTGTAACATTGCCTTGCCCTAGTGAGGACGCTCTCGTGGCTTGTGCTGCTGCCCTGGAGAGAGTCGATCAATCTCCCGTGAAAAACACGGCTTACGCCGAATACGTTGCCTTCGTGACCCGTCAAGACACTGCTGAGACTAAAGACGCTGTGGTCCGTGCTAAACAACAACGTGCTGAGGCCACTGAACGTGTTATGGCTGGCCTGAGAGAGGCTCTGGCTGCTAGAGAACGTCGTGCTCAAATTGAGGCTGAGGGATTGGCAAACCTGAAAACCATGCTCAAAGTCGTGGCTGTACCCGCTACTGTTGCTAAAACTCTCGACCAGGCTCGTAGTGTTGCCGAAATTGCCGATCAAGTCGAAGTGTTGCTGGATCAAACCGAAAAAACTCGTGAACTGGATGTGCCTGCTGTGATCTGGCTCGAACACGCCCAAAGAACATTCGAGACACACCCTTTGTCTGCCGCTCGTGGTGATGGTCCTGGACCCTTGGCTCGTCATGCTGGCCGCCTCGGTGCCCTCTTCGATACTCGTCGTAGAGTAGACGCCTTGAGGAGATCCCTGGAGGAGGCTGAGGCTGAATGGGACGAAGTTTGGGGACGCTTCGGTAGAGTGAGGGGCGGAGCGTGGAAATCTCCGGAGGGATTCCGTGCAATGCATGAGCAACTGAGGGCCCTCCAAGACACAACAAACACCGTGTCTGGCCTGAGGGCTCAACCTGCTTACGAACGCTTGTCTGCTCGCTACCAAGGAGTACTCGGAGCGAAAGGCGCTGAGAGAGCTGAGGCTGTTGAGGAACTCGGTGCTCGTGTCACTAAACACACCGCTCTGTGTGCTAGGCTGAGAGATGAGGTCGTCCGTAGAGTGCCTTGGGAAATGAACTTCGATGCTCTGGGAGGATTGTTGGCTGAGTTCGATGCCGCTGCTGCCGATTTGGCACCTTGGGCTGTAGAGGAATTCCGTGGTGCTAGAGAACTCATTCAATACCGTATGGGCCTGTACTCTGCCTACGCTAGAGCTGGAGGACAAACTGGTGCTGGAGCTGAATCTGCTCCTGCTCCTTTGCTCGTGGATCTGAGGGCTTTGGATGCTCGTGCTCGTGCTTCTTCTTCCCCTGAGGGACATGAAGTGGACCCACAACTGCTGAGGAGGCGTGGAGAGGCTTACTTGAGAGCTGGCGGCGACCCTGGACCTCTCGTGCTCCGTGAAGCTGTTTCTGCTTTGGACCTGCCATTCGCCACATCTTTCTTGGCCCCCGATGGAACTCCCCTCCAATACGCTTTGTGCTTCCCTGCCGTAACGGACAAACTCGGAGCTTTGCTCATGAGGCCCGAGGCCGCTTGTGTTAGACCTCCTTTGCCTACCGATGTGCTGGAATCTGCCCCAACTGTGACTGCCATGTACGTACTCACTGTGGTCAACCGCCTCCAACTGGCATTGAGTGATGCTCAAGCGGCAAACTTCCAACTGTTCGGTCGTTTCGTTCGTCATAGGCAGGCAACCTGGGGAGCGTCAATGGATGCCGCCGCTGAATTGTACGTTGCCCTGGTGGCTACAACTCTCACACGTGAATTCGGTTGTCGCTGGGCACAATTGGGATGGGCTAGTGGAGCTGCTGCTCCTAGACCCCCACCTGGACCCCGTGGCTCACAACGTCACTGTGTGGCATTCAACGAGAACGATGTCCTCGTCGCTTTGGTTGCCGGTGTTCCCGAACACATCTACAACTTCTGGCGCCTGGACTTGGTCCGTCAACACGAGTACATGCACCTCACACTGGAGCGTGCCTTCGAGGATGCTGCCGAGTCTATGCTCTTCGTTCAACGCCTCACTCCACATCCCGACGCTCGTATTAGAGTTCTGCCGACCTTCTTGGATGGTGGTCCTCCTACACGTGGTCTGTTGTTCGGAACCCGCTTGGCGGACTGGCGTCGTGGTAAACTGTCTGAAACCGACCCATTGGCCCCATGGAGATCTGCTTTGGAACTCGGAACCCAACGTCGTGACGTGCCTGCTTTGGGAAAACTGTCCCCTGCTCAAGCTTTGGCCGCTGTGTCGGTACTGGGCCGTATGTGCTTGCCCTCGGCTGCCTTGGCTGCTTTGTGGACCTGTATGTTCCCCGACGACTACACTGAATACGACTCATTCGACGCCCTCTTGGCGGCTCGCCTGGAATCGGGACAAACATTGGGACCTGCTGGCGGTAGAGAGGCTTCATTGTAATAG SEQ ID NO: 44 = construct UL36.4.2.5ATGTCGTACTACCATCACCATCACCATCACGAATACGACTCCTTCGACGCTTTGTTGGCTGCTAGACTGGAATCTGGTCAAACCTTGGGACCCGCTGGCGGTAGAGAGGCTTCTTTGCCCGAGGCTCCTCATGCTTTGTACCGTCCAACCGGACAACATGTTGCTGTGTTGGCGGCTGCTACTCATAGAACCCCTGCTGCTCGTGTTACTGCTATGGACCTGGTCTTGGCGGCCGTTTTGCTGGGCGCTCCTGTGGTGGTCGCTCTGAGAAACACTACTGCCTTCTCCCGTGAATCCGAATTGGAACTGTGCCTCACCCTGTTCGATTCTCGTCCCGGCGGACCGGATGCTGCCCTGAGAGATGTGGTATCCTCCGACATTGAAACCTGGGCTGTGGGCTTGCTCCACACCGATTTGAACCCTATTGAGAACGCTTGCTTGGCGGCTCAACTGCCACGCTTGTCTGCCCTCATTGCTGAACGTCCTTTGGCCGATGGACCCCCTTGTTTGGTGTTGGTGGACATTTCGATGACACCTGTCGCTGTTTTGTGGGAGGCCCCTGAACCACCTGGCCCTCCCGATGTTCGTTTCGTCGGTAGCGAGGCCACTGAGGAATTGCCTTTCGTGGCTACTGCTGGTGATGTTTTGGCGGCGAGTGCTGCCGATGCCGATCCTTTCTTCGCTCGTGCTATCCTGGGCCGTCCTTTCGATGCTTCTCTGCTCACTGGTGAACTGTTCCCTGGTCACCCCGTTTACCAACGTCCCCTGGCGGATGAGGCTGGTCCTTCTGCTCCTACTGCCGCTCGTGATCCTAGAGATCTGGCTGGAGGCGACGGTGGATCCGGACCTGAGGATCCCGCTGCTCCACCTGCTAGACAGGCCGATCCTGGTGTTTTGGCTCCTACTCTGCTCACCGATGCTACTACTGGCGAACCTGTGCCACCCCGTATGTGGGCTTGGATTCATGGACTGGAGGAACTGGCTTCCGATGATGCCGGCGGTCCTACCCCAAACCCTGCCCCGGCTTTGCTGCCCCCTCCTGCTACGGATCAATCTGTCCCCACTTCCCAATACGCCCCTAGACCAATTGGCCCGGCTGCCACTGCTAGAGAAACTCGTCCTTCCGTTCCCCCTCAACAAAACACTGGTCGTGTCCCTGTGGCTCCACGTGATGACCCTAGACCTTCCCCCCCTACTCCTTCCCCCCCTGCCGATGCTGCTTTGCCACCTCCTGCCTTCTCTGGTTCTGCTGCTGCTTTCTCCGCTGCTGTTCCACGTGTTCGTCGTTCTAGGCGTACTCGTGCCAAATCCCGTGCCCCTCGTGCTTCTGCCCCACCCGAGGGATGGCGTCCCCCCGCTTTGCCTGCCCCTGTTGCTCCTGTGGCGGCTTCTGCTCGTCCCCCCGATCAACCTCCTACTCCCGAATCTGCTCCCCCGGCTTGGGTTTCCGCTCTGCCATTGCCACCCGGACCTGCTAGTGCTCGTGGTGCTTTCCCTGCTCCAACCTTGGCCCCTATTCCCCCACCCCCCGCTGAGGGAGCTGTTGTTCCCGGTGGTGATCGTAGACGTGGTCGCCGTCAAACAACTGCTGGACCATCCCCTACACCGCCACGTGGCCCGGCTGCTGGTCCTCCTCGTCGCCTCACTAGGCCTGCTGTTGCTAGTCTGTCCGCTTCTTTGAACTCTCTGCCTTCCCCCCGTGATCCTGCCGATCATGCTGCTGCCGTTTCTGCTGCCGCCGCTGCCGTACCACCTTCACCTGGACTGGCTCCCCCAACTTCTGCTGTCCAAACCTCTCCTCCTCCCTTGGCGCCTGGTCCTGTTGCCCCATCTGAACCTTTGTGTGGCTGGGTTGTGCCTGGAGGCCCTGTTGCTAGACGTCCCCCACCCCAATCTCCGGCTACTAAACCGGCTGCTCGTACCCGTATTAGGGCTCGTTCTGTGCCCCAACCACCCTTGCCCCAACCTCCACTGCCTCAACCCCCCTTGCCTCAACCCCCTCTCCCCCAACCACCTCTGCCTCAACCTCCGCTGCCCCAACCTCCTTTGCCCCAACCTCCTTTGCCCCAACCTCCTTTGCCCCAACCTCCGCTGCCCCAACCTCCGCTGCCACCTGTTACTCGTACACTCACTCCCCAATCTCGTGACTCTGTGCCTACACCTGAGTCTCCAACTCACACAAACACCCACTTGCCCGTTAGTGCTGTGACTTCTTGGGCTTCGTCCCTGGCTCTCCATGTGGATTCTGCCCCTCCCCCTGCTTCATTGCTCCAAACTCTCCACATTTCCTCCGATGATGAACACTCCGACGCCGACTCACTCCGCTTCTCCGATTCCGATGACACTGAGGCTCTCGATCCTTTGCCTCCTGAACCTCACTTGCCACCTGCCGATGAACCCCCCGGACCTCTGGCTGCCGACCATCTCCAATCACCTCACTCACAATTCGGTCCTTTGCCCGTTCAAGCGAACGCTGTTCTGTCTCGTCGTTACGTGAGATCAACTGGCCGTTCTGCCTTGGCTGTGCTCATTAGAGCTTGTCGCCGTATCCAACAACAACTCCAGCGTACTAGGAGAGCACTCTTCCAACGCTCAAACGCCGTGCTCACATCACTCCACCATGTCCGTATGCTCTTGGGATAATAG SEQ ID NO: 45 = US12ATGTCTTGGGCTCTGAAAACCACCGACATGTTCCTGGACTCTTCTCGTTGCACCCACCGTACCTACGGTGACGTTTGCGCTGAAATCCACAAACGTGAACGTGAAGACCGTGAAGCTGCTCGTACCGCTGTTACCGACCCGGAACTGCCGCTGCTGTGCCCGCCGGACGTTCGTTCTGACCCGGCTTCTCGTAACCCGACCCAGCAGACCCGTGGTTGCGCTCGTTCTAACGAACGTCAGGACCGTGTTCTGGCTCCGTGA SEQ ID NO: 46 = US4ATGAAGTTCCTCGTGAACGTGGCCCTGGTGTTCATGGTGGTGTACATCAGCTACATCTACGCTAACCGTTGGGGTTCCGGCGTGCCCGGTCCCATCAACCCCCCCAACTCCGACGTGGTGTTCCCCGGTGGTTCCCCCGTGGCTCAGTACTGCTACGCTTACCCCCGTCTGGACGACCCTGGTCCCCTGGGTTCTGCTGACGCTGGTCGTCAGGACCTGCCCCGTCGTGTCGTGCGTCACGAGCCCCTGGGTCGTAGCTTCCTGACCGGTGGCCTGGTGCTGTTGGCTCCCCCTGTGCGCGGTTTCGGTGCTCCCAACGCTACCTACGCTGCTCGTGTGACCTACTACCGTCTGACCCGTGCTTGCCGTCAGCCCATCCTGCTGCGTCAGTACGGTGGTTGCCGTGGTGGAGAGCCCCCATCCCCCAAGACCTGCGGTTCTTACACCTACACCTACCAGGGTGGTGGTCCCCCTACCCGTTACGCTCTGGTCAACGCTTCCCTGCTGGTGCCCATCTGGGACCGTGCTGCTGAGACTTTCGAGTACCAGATCGAGCTGGGTGGCGAGCTGCACGTGGGTCTGCTGTGGGTGGAAGTGGGTGGAGAGGGTCCCGGTCCTACCGCTCCTCCTCAGGCTGCTCGTGCTGAGGGTGGTCCTTGCGTGCCACCCGTGCCTGCTGGTCGTCCTTGGCGTTCCGTGCCCCCCGTGTGGTACTCCGCTCCCAACCCCGGTTTCCGCGGTCTGCGTTTCCGTGAGCGTTGCCTGCCTCCCCAGACCCCTGCTGCTCCTTCCGACCTGCCTCGTGTGGCTTTCGCTCCCCAGTCCCTGCTCGTGGGTATCACCGGTCGTACCTTCATCCGTATGGCTCGTCCCACCGAGGACGTGGGTGTCCTGCCTCCTCACTGGGCTCCAGGTGCTCTGGACGACGGTCCCTACGCTCCCTTCCCCCCTCGTCCCCGTTTCCGTCGTCACCACCACCATCACCACTAATAASEQ ID NO: 117 = construct RS1.2ATGTCGTACTACCATCACCATCACCATCACATGGTGCTGTACGGCGGGCTGGGCGACAGCCGCCCCGGCCTCTGGGGGGCGCCCGAGGCGGAGGAGGCGCGGGCCCGGTTCGAGGCCTCGGGCGCCCCGGCGCCCGTGTGGGCGCCCGAGCTGGGCGACGCGGCGCAGCAGTACGCCCTGATCACGCGGCTGCTGTACACGCCGGACGCGGAGGCGATGGGGTGGCTCCAGAACCCGCGCGTGGCGCCCGGGGACGTGGCGCTGGACCAGGCCTGCTTCCGGATCTCGGGCGCGGCGCGCAACAGCAGCTCCTTCATCTCCGGCAGCGTGGCGCGGGCCGTGCCCCACCTGGGGTACGCCATGGCGGCGGGCCGCTTCGGCTGGGGCCTGGCGCACGTGGCGGCCGCCGTGGCCATGAGCCGCCGCTACGACCGCGCGCAGAAGGGCTTCCTGCTGACCAGCCTGCGCCGCGCCTACGCGCCCCTGCTGGCGCGCGAGAACGCGGCGCTGACCGGGGCGCGGACCCCCGACGACGGCGGCGACGCCAACCGCCGCGACGGCGACGACGCCCGCGGGAAGCCCGCCGCCGCCGCCGCCCCGTTGCCGTCGGCGGCGGCGTCGCCGGCCGACGAGCGCGCGGTGCCCGCCGGCTACGGCGCCGCGGGGGTGCTCGCCGCCCTGGGGCGCCTGAGCGCCGCGCCCGCCTCCGCGCCGGCCGGGGCCGACGACGACGACGACGACGACGACGGCGCCGGCGGTGGTGGCGGTGGTGGCGGTGGTGGCGGCGGCCGGCGCGCGGAGGCGGGCCGCGTGGCCGTGGAGTGCCTGGCCGCCTGCCGCGGGATCCTGGAGGCGCTGGCGGAGGGCTTCGACGGCGACCTGGCGGCCGTGCCGGGGCTGGCCGGAGCCCGGCCCGCCGCGCCCCCGCGCCCGGGGCCCGCGGGCGCGGCCGCCCCGCCGCACGCCGACGCGCCCCGCCTGCGCGCCTGGCTGCGCGAGCTGCGGTTCGTGCGCGACGCGCTGGTGCTGATGCGCCTGCGCGGGGACCTGCGCGTGGCCGGCGGCAGCGAGGCCGCCGTGGCCGCCGTGCGCGCCGTGAGCCTGGTCGCCGGGGCCCTGGGCCCGGCGCTGCCGCGGAGCCCGCGCCTGCTGAGCTCCGCCGCCGCCGCCGCCGCGGACCTGCTCTTCCAGAACCAGAGCCTGAGTACTAGAGGATCATAASEQ ID NO: 118 = UL1ATGTCGTACTACCATCACCATCACCATCACATGGGGTTCGTCTGTCTGTTTGGGCTTGTCGTTATGGGAGCCTGGGGGGCGTGGGGTGGGTCACAGGCAACCGAATATGTTCTTCGTAGTGTTATTGCCAAAGAGGTGGGGGACATACTAAGAGTGCCTTGCATGCGGACCCCCGCGGACGATGTTTCTTGGCGCTACGAGGCCCCGTCCGTTATTGACTATGCCCGCATAGACGGAATATTTCTTCGCTATCACTGCCCGGGGTTGGACACGTTTTTGTGGGATAGGCACGCCCAGAGGGCGTATCTTGTTAACCCCTTTCTCTTTGCGGCGGGATTTTTGGAGGACTTGAGTCACTCTGTGTTTCCGGCCGACACCCAGGAAACAACGACGCGCCGGGCCCTTTATAAAGAGATACGCGATGCGTTGGGCAGTCGAAAACAGGCCGTCAGCCACGCACCCGTCAGGGCCGGGTGTGTAAACTTTGACTACTCACGCACTCGCCGCTGCGTCGGGCGACGCGATTTACGGCCTGCCAACACCACGTCAACGTGGGAACCGCCTGTGTCGTCGGACGATGAAGCGAGCTCGCAGTCGAAGCCCCTCGCCACCCAGCCGCCCGTCCTCGCCCTTTCGAACGCCCCCCCACGGCGGGTCTCCCCGACGCGAGGTCGGCGCCGGCATACTCGCCTCCGACGCAACTGA SEQ ID NO: 119 = construct UL1sATGAAGTTCCTCGTGAACGTGGCCCTGGTGTTCATGGTGGTGTACATCAGCTACATCTACGCCAACCGTTGGGGGTTCGTCTGTCTGTTTGGGCTTGTCGTTATGGGAGCCTGGGGGGCGTGGGGTGGGTCACAGGCAACCGAATATGTTCTTCGTAGTGTTATTGCCAAAGAGGTGGGGGACATACTAAGAGTGCCTTGCATGCGGACCCCCGCGGACGATGTTTCTTGGCGCTACGAGGCCCCGTCCGTTATTGACTATGCCCGCATAGACGGAATATTTCTTCGCTATCACTGCCCGGGGTTGGACACGTTTTTGTGGGATAGGCACGCCCAGAGGGCGTATCTTGTTAACCCCTTTCTCTTTGCGGCGGGATTTTTGGAGGACTTGAGTCACTCTGTGTTTCCGGCCGACACCCAGGAAACAACGACGCGCCGGGCCCTTTATAAAGAGATACGCGATGCGTTGGGCAGTCGAAAACAGGCCGTCAGCCACGCACCCGTCAGGGCCGGGTGTGTAAACTTTGACTACTCACGCACTCGCCGCTGCGTCGGGCGACGCGATTTACGGCCTGCCAACACCACGTCAACGTGGGAACCGCCTGTGTCGTCGGACGATGAAGCGAGCTCGCAGTCGAAGCCCCTCGCCACCCAGCCGCCCGTCCTCGCCCTTTCGAACGCCCCCCCACGGCGGGTCTCCCCGACGCGAGGTCGGCGCCGGCATACTCGCCTCCGACGCAACCATCACCATCACCATCACTGASEQ ID NO: 120 = construct UL19ΔTEVATGTCGTACTACCATCACCATCACCATCACATGGCCGCTCCTGCCCGCGACCCCCCGGGTTACCGGTACGCCGCGGCCATGGTGCCCACCGGCTCCATCCTGAGTACGATCGAGGTGGCGTCCCACCGCAGACTCTTTGATTTTTTCGCCCGCGTGCGCTCCGACGAAAACAGCCTGTATGACGTAGAGTTTGACGCCCTGCTGGGGTCCTACTGCAACACCCTGTCGCTCGTGCGCTTTCTGGAGCTCGGCCTGTCCGTGGCGTGCGTGTGCACCAAGTTCCCGGAGCTGGCTTACATGAACGAAGGGCGTGTGCAGTTCGAGGTCCACCAGCCCCTCATCGCCCGCGACGGCCCGCACCCCGTCGAGCAGCCCGTGCATAATTACATGACGAAGGTCATCGACCGCCGGGCCCTGAACGCCGCCTTCAGCCTGGCCACCGAGGCCATTGCCCTGCTCACGGGGGAGGCCCTGGACGGGACGGGCATTAGCCTGCATCGCCAGCTGCGCGCCATCCAGCAGCTCGCGCGCAACGTCCAGGCCGTCCTGGGGGCGTTTGAGCGCGGCACGGCCGACCAGATGCTGCACGTGCTGTTGGAGAAGGCGCCTCCCCTGGCCCTGCTGTTGCCCATGCAACGATATCTCGACAACGGGCGCCTGGCGACCAGGGTTGCCCGGGCGACCCTGGTCGCCGAGCTGAAGCGGAGCTTTTGCGACACGAGCTTCTTCCTGGGCAAGGCGGGCCATCGCCGCGAGGCCATCGAGGCCTGGCTCGTGGACCTGACCACGGCGACGCAGCCGTCCGTGGCCGTGCCCCGCCTGACGCACGCCGACACGCGCGGGCGGCCGGTCGACGGGGTGCTGGTCACCACCGCCGCCATCAAACAGCGCCTCCTGCAGTCCTTCCTGAAGGTGGAGGACACCGAGGCCGACGTGCCGGTGACCTACGGCGAGATGGTCTTGAACGGGGCCAACCTCGTCACGGCGCTGGTGATGGGCAAGGCCGTGCGGAGCCTGGACGACGTGGGCCGCCACCTGCTGGAGATGCAGGAGGAGCAACTCGAGGCGAACCGGGAGACGCTGGATGAACTCGAAAGCGCCCCCCAGACAACGCGCGTGCGCGCGGATCTGGTGGCCATAGGCGACAGGCTGGTCTTCCTGGAGGCCCTGGAGAAGCGCATCTACGCCGCCACCAACGTGCCCTACCCCCTGGTGGGCGCCATGGACCTGACGTTCGTCCTGCCCCTGGGGCTGTTCAACCCGGCCATGGAGCGCTTCGCCGCGCACGCCGGGGACCTGGTGCCCGCCCCCGGCCACCCGGAGCCCCGCGCGTTCCCTCCCCGGCAGCTGTTTTTTTGGGGAAAGGACCACCAGGTTCTGCGGCTGTCCATGGAGAACGCGGTCGGGACCGTGTGTCATCCTTCGCTCATGAACATCGACGCGGCCGTCGGGGGCGTGAACCACGACCCCGTCGAGGCCGCGAATCCGTACGGGGCGTACGTCGCGGCCCCGGCCGGCCCCGGCGCGGACATGCAGCAGCGTTTTCTGAACGCCTGGCGGCAGCGCCTCGCCCACGGCCGGGTCCGGTGGGTCGCCGAGTGCCAGATGACCGCGGAGCAGTTCATGCAGCCCGACAACGCCAACCTGGCTCTGGAGCTGCACCCCGCGTTCGACTTCTTCGCGGGCGTGGCCGACGTCGAGCTTCCCGGCGGCGAAGTCCCCCCGGCCGGTCCGGGGGCGATCCAGGCCACCTGGCGCGTGGTCAACGGCAACCTGCCCCTGGCGCTGTGTCCGGTGGCGTTTCGTGACGCCCGGGGCCTGGAGCTCGGCGTTGGCCGCCACGCCATGGCGCCGGCTACCATAGCCGCCGTCCGCGGGGCGTTCGAGGACCGCAGCTACCCGGCGGTGTTCTACCTGCTGCAAGCCGCGATTCACGGCAGCGAGCACGTGTTCTGCGCCCTGGCGCGGCTCGTGACTCAGTGCATCACCAGCTACTGGAACAACACGCGATGCGCGGCGTTCGTGAACGACTACTCGCTGGTCTCGTACATCGTGACCTACCTCGGGGGCGACCTCCCCGAGGAGTGCATGGCCGTGTATCGGGACCTGGTGGCCCACGTCGAGGCCCTGGCCCAGCTGGTGGACGACTTTACCCTGCCGGGCCCGGAGCTGGGCGGGCAGGCTCAGGCCGAGCTGAATCACCTGATGCGCGACCCGGCGCTGCTGCCGCCCCTCGTGTGGGACTGCGACGGCCTTATGCGACACGCGGCCCTGGACCGCCACCGAGACTGCCGGATTGACGCGGGGGAGCACGAGCCCGTCTACGCGGCGGCGTGCAACGTGGCGACGGCCGACTTTAACCGCAACGACGGCCGGCTGCTGCACAACACCCAGGCCCGCGCGGCCGACGCCGCCGACGACCGGCCGCACCGGCCGGCCGACTGGACCGTCCACCACAAAATCTACTATTACGTGCTGGTGCCGGCCTTCTCGCGGGGGCGCTGCTGCACCGCGGGGGTCCGCTTCGACCGCGTGTACGCCACGCTGCAGAACATGGTGGTCCCGGAGATCGCCCCCGGCGAGGAGTGCCCGAGCGATCCCGTGACCGACCCCGCCCACCCGCTGCATCCCGCCAATCTGGTGGCCAACACGGTCAACGCCATGTTCCACAACGGGCGCGTCGTCGTCGACGGGCCCGCCATGCTCACGCTGCAGGTGCTGGCGCACAACATGGCCGAGCGCACGACGGCGCTGCTGTGCTCCGCGGCGCCCGACGCGGGCGCCAACACCGCGTCGACGGCCAACATGCGCATCTTCGACGGGGCGCTGCACGCCGGCGTGCTGCTCATGGCCCCCCAGCACCTGGACCACACCATCCAAAATGGCGAATACTTCTACGTCCTGCCCGTCCACGCGCTGTTTGCGGGCGCCGACCACGTGGCCAACGCGCCCAACTTCCCCCCGGCCCTGCGCGACCTGGCGCGCCACGTCCCCCTGGTCCCCCCGGCCCTGGGGGCCAACTACTTCTCCTCCATCCGCCAGCCCGTGGTGCAGCACGCCCGCGAGAGCGCGGCGGGGGAGAACGCGCTGACCTACGCGCTCATGGCGGGGTACTTCAAGATGAGCCCCGTGGCCCTGTATCACCAGCTCAAGACGGGCCTCCACCCCGGGTTCGGGTTCACCGTCGTGCGGCAGGACCGCTTCGTGACCGAGAACGTGCTGTTTTCCGAGCGCGCGTCGGAGGCGTACTTTCTGGGCCAGCTCCAGGTGGCCCGCCACGAAACGGGCGGGGGGGTCAGCTTCACGCTCACCCAGCCGCGCGGAAACGTGGACCTGGGTGTGGGCTACACCGCCGTCGCGGCCACGGCCACCGTCCGCAACCCCGTTACGGACATGGGCAACCTCCCCCAAAACTTTTACCTCGGCCGCGGGGCCCCCCCGCTGCTAGACAACGCGGCCGCCGTGTACCTGCGCAACGCGGTCGTGGCGGGAAACCGGCTGGGGCCGGCCCAGCCCCTCCCGGTCTTTGGCTGCGCCCAGGTGCCGCGGCGCGCCGGCATGGACCACGGGCAGGATGCCGTGTGTGAGTTCATCGCCACCCCCGTGGCCACGGACATCAACTACTTTCGCCGGCCCTGCAACCCGCGGGGACGCGCGGCCGGCGGCGTGTACGCGGGGGACAAGGAGGGGGACGTCATAGCCCTCATGTACGACCACGGCCAGAGCGACCCGGCGCGGCCCTTCGCGGCCACGGCCAACCCGTGGGCGTCGCAGCGGTTCTCGTACGGGGACCTGCTGTACAACGGGGCCTATCACCTCAACGGGGCCTCGCCCGTCCTCAGCCCCTGCTTCAAGTTCTTCACCGCGGCCGACATCACGGCCAAACATCGCTGCCTGGAGCGTCTTATCGTGGAAACGGGATCGGCGGTATCCACGGCCACCGCTGCCAGCGACGTGCAGTTTAAGCGCCCGCCGGGGTGCCGCGAGCTCGTGGAAGACCCGTGCGGCCTGTTTCAGGAAGCCTACCCGATCACCTGCGCCAGCGACCCCGCCCTGCTACGCAGCGCCCGCGATGGGGAGGCCCACGCGCGAGAGACCCACTTTACGCAGTATCTCATCTACGACGCCTCCCCGCTAAAGGGCCTGTCTCTGTAASEQ ID NO: 121 = construct RS1.1ATGAGTGCCGAACAGCGTAAAAAGAAAAAAACCACCACCACGACCCAAGGACGTGGAGCTGAAGTTGCTATGGCGGATGAGGATGGAGGCCGCTTGAGAGCTGCTGCTGAGACTACTGGAGGACCTGGATCACCGGACCCTGCCGATGGACCCCCCCCTACACCAAACCCCGATCGTAGACCGGCTGCTAGACCTGGATTCGGATGGCATGGAGGACCCGAGGAAAACGAGGACGAGGCGGACGACGCCGCTGCCGACGCCGACGCCGATGAGGCTGCCCCTGCTTCTGGAGAGGCGGTAGACGAACCTGCTGCCGATGGAGTTGTTAGCCCTAGGCAATTGGCTTTGTTGGCGAGCATGGTAGACGAGGCTGTGAGAACAATCCCTTCCCCTCCCCCTGAACGTGATGGAGCACAAGAGGAGGCGGCTAGGAGTCCCTCACCACCCCGTACACCTTCTATGAGAGCGGATTACGGCGAGGAAAACGACGACGACGACGATGATGATGACGACGATGATCGTGATGCCGGACGCTGGGTTAGGGGACCTGAAACCACTTCTGCTGTCCGTGGAGCATACCCCGATCCTATGGCGAGTTTGAGCCCTAGACCACCTGCCCCGAGGAGACACCACCACCACCACCATCATAGGCGTAGACGTGCTCCTAGACGTCGTTCTGCCGCTAGTGACTCTTCCAAATCTGGCTCTTCTTCATCTGCCTCTTCCGCTTCATCTTCGGCCTCATCGTCCTCTTCGGCATCCGCTTCGAGTAGTGATGATGATGATGACGACGACGCTGCTAGAGCCCCCGCTTCTGCTGCCGACCACGCTGCTGGCGGAACTTTGGGAGCCGACGACGAGGAGGCGGGAGTTCCTGCTCGTGCCCCGGGAGCTGCTCCGAGGCCTTCTCCACCCCGTGCTGAACCTGCTCCGGCTAGAACACCGGCCGCTACTGCTGGTAGACTGGAGCGTAGACGTGCCCGTGCTGCTGTGGCTGGTAGAGATGCTACTGGCCGCTTCACTGCTGGCCGTCCTAGACGTGTTGAACTGGACGCCGATGCTGCTTCTGGTGCTTTCTACGCCCGTTACCGTGATGGTTACGTGTCTGGTGAACCTTGGCCTGGCGCTGGTCCACCTCCGCCCGGACGTGTACTCTACGGTGGATTGGGCGATTCTCGCCCTGGTCTGTGGGGCGCTCCG SEQ ID NO: 122 =construct RS1.3.1TCGAGTGCCGCCGCTGCTGCCGCCGATTTGTTGTTCCAAAACCAATCCCTCCGCCCTCTGCTCGCCGACACTGTTGCCGCTGCCGATTCTCTGGCTGCTCCGGCTTCTGCCCCACGTGAAGCTCGTAAACGTAAATCACCCGCTCCGGCTCGTGCTCCCCCTGGTGGCGCCCCTAGACCCCCTAAAAAATCCCGTGCCGATGCCCCTAGACCTGCTGCTGCTCCCCCCGCTGGTGCTGCTCCCCCCGCTCCCCCTACTCCCCCCCCACGCCCACCTCGTCCCGCTGCCCTCACACGCCGTCCTGCTGAGGGACCCGATCCACAAGGCGGCTGGCGTAGACAACCTCCTGGCCCATCCCATACACCGGCACCATCTGCCGCTGCTTTGGAGGCTTACTGTGCTCCTCGTGCTGTGGCTGAACTCACCGATCATCCGCTGTTCCCTGCTCCCTGGCGTCCCGCCCTCATGTTCGATCCTAGAGCTTTGGCTTCCTTGGCCGCTCGTTGTGCTGCCCCTCCCCCTGGCGGTGCTCCGGCTGCTTTCGGTCCTCTCCGTGCCTCTGGTCCACTCCGCCGTGCCGCTGCCTGGATGAGACAAGTTCCCGACCCTGAGGATGTTAGAGTTGTGATCTTGTACTCGCCCTTGCCTGGCGAGGATTTGGCCGCTGGTAGAGCTGGCGGTGGCCCCCCTCCTGAATGGTCTGCTGAACGTGGTGGTTTGTCTTGCTTGTTGGCCGCCCTGGGAAACCGTCTGTGTGGTCCTGCTACTGCTGCTTGGGCTGGAAACTGGACTGGCGCTCCCGATGTTTCTGCTCTCGGTGCTCAA SEQ ID NO: 123 =construct RS1.3.2TGGGCTGGAAACTGGACTGGCGCTCCCGATGTTTCTGCTCTCGGTGCTCAAGGAGTTTTGCTGCTCTCTACTCGTGACTTGGCATTCGCTGGAGCTGTTGAATTCCTGGGACTCTTGGCTGGCGCTTGTGATAGGAGACTCATCGTCGTAAACGCTGTGAGAGCTGCCGATTGGCCTGCCGATGGTCCTGTTGTGTCTCGTCAACACGCTTACTTGGCTTGTGAAGTGTTGCCCGCTGTCCAATGTGCTGTTCGCTGGCCTGCTGCTCGTGATCTGAGGCGTACTGTTCTGGCTAGTGGTCGTGTTTTCGGACCTGGTGTTTTCGCTCGTGTCGAAGCTGCTCACGCTAGACTGTACCCCGATGCCCCACCCCTCCGTTTGTGTCGTGGAGCAAACGTTCGCTACCGTGTCCGTACTCGTTTCGGACCCGATACTCTGGTTCCAATGTCCCCTCGTGAATACCGTCGTGCTGTTCTGCCTGCCCTCGATGGACGTGCTGCCGCTTCTGGCGCTGGTGACGCTATGGCTCCTGGCGCTCCGGACTTCTGTGAGGATGAGGCTCACTCACATCGTGCCTGTGCCCGCTGGGGACTGGGCGCTCCATTGAGGCCTGTATACGTGGCACTGGGCCGTGATGCTGTTAGAGGCGGACCCGCTGAATTGAGAGGCCCTCGTCGTGAATTCTGTGCTAGGGCTCTGCTCGAACCCGATGGAGATGCTCCTCCTTTGGTACTCCGTGACGACGCCGATGCTGGTCCTCCCCCACAAATTCGCTGGGCTAGTGCTGCTGGACGTGCTGGTACTGTATTGGCTGCTGCTGGCGGTGGCGTTGAAGTTGTTGGTACTGCCGCTGGACTCGCTACACCTCCCCGCCGTGAACCTGTAGACATGGATGCTGAACTCGAGGATGATGACGACGGATTGTTCGGAGAG SEQ ID NO: 124 = construct RS1.3TCGAGTGCCGCCGCTGCTGCCGCCGATTTGTTGTTCCAAAACCAATCCCTCCGCCCTCTGCTCGCCGACACTGTTGCCGCTGCCGATTCTCTGGCTGCTCCGGCTTCTGCCCCACGTGAAGCTCGTAAACGTAAATCACCCGCTCCGGCTCGTGCTCCCCCTGGTGGCGCCCCTAGACCCCCTAAAAAATCCCGTGCCGATGCCCCTAGACCTGCTGCTGCTCCCCCCGCTGGTGCTGCTCCCCCCGCTCCCCCTACTCCCCCCCCACGCCCACCTCGTCCCGCTGCCCTCACACGCCGTCCTGCTGAGGGACCCGATCCACAAGGCGGCTGGCGTAGACAACCTCCTGGCCCATCCCATACACCGGCACCATCTGCCGCTGCTTTGGAGGCTTACTGTGCTCCTCGTGCTGTGGCTGAACTCACCGATCATCCGCTGTTCCCTGCTCCCTGGCGTCCCGCCCTCATGTTCGATCCTAGAGCTTTGGCTTCCTTGGCCGCTCGTTGTGCTGCCCCTCCCCCTGGCGGTGCTCCGGCTGCTTTCGGTCCTCTCCGTGCCTCTGGTCCACTCCGCCGTGCCGCTGCCTGGATGAGACAAGTTCCCGACCCTGAGGATGTTAGAGTTGTGATCTTGTACTCGCCCTTGCCTGGCGAGGATTTGGCCGCTGGTAGAGCTGGCGGTGGCCCCCCTCCTGAATGGTCTGCTGAACGTGGTGGTTTGTCTTGCTTGTTGGCCGCCCTGGGAAACCGTCTGTGTGGTCCTGCTACTGCTGCTTGGGCTGGAAACTGGACTGGCGCTCCCGATGTTTCTGCTCTCGGTGCTCAAGGAGTTTTGCTGCTCTCTACTCGTGACTTGGCATTCGCTGGAGCTGTTGAATTCCTGGGACTCTTGGCTGGCGCTTGTGATAGGAGACTCATCGTCGTAAACGCTGTGAGAGCTGCCGATTGGCCTGCCGATGGTCCTGTTGTGTCTCGTCAACACGCTTACTTGGCTTGTGAAGTGTTGCCCGCTGTCCAATGTGCTGTTCGCTGGCCTGCTGCTCGTGATCTGAGGCGTACTGTTCTGGCTAGTGGTCGTGTTTTCGGACCTGGTGTTTTCGCTCGTGTCGAAGCTGCTCACGCTAGACTGTACCCCGATGCCCCACCCCTCCGTTTGTGTCGTGGAGCAAACGTTCGCTACCGTGTCCGTACTCGTTTCGGACCCGATACTCTGGTTCCAATGTCCCCTCGTGAATACCGTCGTGCTGTTCTGCCTGCCCTCGATGGACGTGCTGCCGCTTCTGGCGCTGGTGACGCTATGGCTCCTGGCGCTCCGGACTTCTGTGAGGATGAGGCTCACTCACATCGTGCCTGTGCCCGCTGGGGACTGGGCGCTCCATTGAGGCCTGTATACGTGGCACTGGGCCGTGATGCTGTTAGAGGCGGACCCGCTGAATTGAGAGGCCCTCGTCGTGAATTCTGTGCTAGGGCTCTGCTCGAACCCGATGGAGATGCTCCTCCTTTGGTACTCCGTGACGACGCCGATGCTGGTCCTCCCCCACAAATTCGCTGGGCTAGTGCTGCTGGACGTGCTGGTACTGTATTGGCTGCTGCTGGCGGTGGCGTTGAAGTTGTTGGTACTGCCGCTGGACTCGCTACACCTCCCCGCCGTGAACCTGTAGACATGGATGCTGAACTCGAGGATGATGACGACGGATTGTTCGGAGAG SEQ ID NO: 125 = construct RS1.4ACTGCTGGCCGTCCTAGACGTGTTGAACTGGACGCCGATGCTGCTTCTGGTGCTTTCTACGCCCGTTACCGTGATGGTTACGTGTCTGGTGAACCTTGGCCTGGCGCTGGTCCACCTCCGCCCGGACGTGTACTCTACGGTGGATTGGGCGATTCTCGCCCTGGTCTGTGGGGCGCTCCGGAGGCTGAGGAGGCTAGAGCCCGTTTCGAGGCTTCTGGTGCCCCTGCTCCTGTTTGGGCTCCTGAATTGGGCGACGCTGCTCAACAATACGCCCTCATCACACGCTTGCTGTACACTCCCGACGCCGAGGCTATGGGATGGCTCCAAAACCCTAGAGTTGCCCCTGGTGATGTTGCTCTGGATCAGGCTTGTTTCCGTATCTCCGGCGCTGCTCGTAACTCTTCTTCGTTCATCTCCGGTTCTGTGGCTAGAGCTGTGCCTCACTTGGGATACGCCATGGCCGCTGGACGTTTCGGCTGGGGACTGGCTCATGTTGCTGCCGCTGTAGCAATGTCTAGACGCTACGACCGTGCTCAAAAAGGATTCTTGCTCACGTCACTGAGGCGTGCTTACGCCCCTTTGTTGGCCCGTGAAAACGCTGCCCTCACTGGCGCCCGTACCCCCGATGACGGTGGCGACGCCAACCGCCACGATGGTGATGATGCTAGAGGCAAACCCGCTGCCGCTGCTGCTCCTTTGCCCTCTGCCGCCGCTTCCCCTGCCGATGAACGTGCTGTTCCTGCCGGTTACGGTGCCGCTGGTGTGTTGGCTGCTTTGGGACGCTTGAGTGCTGCCCCGGCTAGTGCCCCCGCTGGTGCCGATGACGATGACGATGACGATGGTGCTGGCGGAGGCGGTGGCGGTAGACGTGCTGAGGCTGGACGTGTTGCTGTTGAATGCCTGGCTGCCTGTAGAGGAATCTTGGAGGCTCTGGCCGAGGGATTCGACGGAGACTTGGCGGCTGTACCGGGACTGGCGGGAGCGAGGCCTGCCGCTCCACCTCGCCCCGGTCCTGCTGGTGCTGCCGCTCCTCCTCATGCCGACGCTCCTAGACTCCGTGCTTGGCTCCGTGAACTCCGTTTCGTTCGTGACGCTTTGGTTCTGATGAGACTGAGAGGCGACTTGAGAGTGGCTGGAGGATCCGAGGCTGCTGTTGCTGCTGTCCGTGCTGTTTCTTTGGTTGCTGGTGCTTTGGGCCCTGCTTTGCCGAGATCTCCCCGTTTGTTGTCGAGTGCCGCCGCTGCTGCCGCCGATTTGTTGTTCCAAAACCAATCCCTCCGCCCTCTGCTCGCCGACACTGTTGCCGCTGCCGATTCTCTGGCTGCTCCGGCTTCTGCCCCACGTGAAGCTCGTAAACGTAAATCACCCGCTCCGGCTCGTGCTCCCCCTGGTGGCGCCCCTAGACCCCCTAAAAAATCCCGTGCCGATGCCCCTAGACCTGCTGCTGCTCCCCCCGCTGGTGCTGCTCCCCCCGCTCCCCCTACTCCCCCCCCACGCCCACCTCGTCCCGCTGCCCTCACACGCCGTCCTGCTGAGGGACCCGATCCACAAGGCGGCTGGCGTAGACAACCTCCTGGCCCATCCCATACACCGGCACCATCTGCCGCTGCTTTGGAGGCTTACTGTGCT SEQ ID NO: 126 = construct RS1.5GCCGCTGCCGATTCTCTGGCTGCTCCGGCTTCTGCCCCACGTGAAGCTCGTAAACGTAAATCACCCGCTCCGGCTCGTGCTCCCCCTGGTGGCGCCCCTAGACCCCCTAAAAAATCCCGTGCCGATGCCCCTAGACCTGCTGCTGCTCCCCCCGCTGGTGCTGCTCCCCCCGCTCCCCCTACTCCCCCCCCACGCCCACCTCGTCCCGCTGCCCTCACACGCCGTCCTGCTGAGGGACCCGATCCACAAGGCGGCTGGCGTAGACAACCTCCTGGCCCATCCCATACACCGGCACCATCTGCCGCTGCTTTGGAGGCTTACTGTGCTCCTCGTGCTGTGGCTGAACTCACCGATCATCCGCTGTTCCCTGCTCCCTGGCGTCCCGCCCTCATGTTCGATCCTAGAGCTTTGGCTTCCTTGGCCGCTCGTTGTGCTGCCCCTCCCCCTGGCGGTGCTCCGGCTGCTTTCGGTCCTCTCCGTGCCTCTGGTCCACTCCGCCGTGCCGCTGCCTGGATGAGACAAGTTCCCGACCCTGAGGATGTTAGAGTTGTGATCTTGTACTCGCCCTTGCCTGGCGAGGATTTGGCCGCTGGTAGAGCTGGCGGTGGCCCCCCTCCTGAATGGTCTGCTGAACGTGGTGGTTTGTCTTGCTTGTTGGCCGCCCTGGGAAACCGTCTGTGTGGTCCTGCTACTGCTGCTTGGGCTGGAAACTGGACTGGCGCTCCCGATGTTTCTGCTCTCGGTGCTCAAGGAGTTTTGCTGCTCTCTACTCGTGACTTGGCATTCGCTGGAGCTGTTGAATTCCTGGGACTCTTGGCTGGCGCTTGTGATAGGAGACTCATCGTCGTAAACGCTGTGAGAGCTGCCGATTGGCCTGCCGATGGTCCTGTTGTGTCTCGTCAACACGCTTACTTGGCTTGTGAAGTGTTGCCCGCTGTCCAATGTGCTGTTCGCTGGCCTGCTGCTCGTGATCTGAGGCGTACTGTTCTGGCTAGTGGTCGTGTTTTCGGACCTGGTGTTTTCGCTCGTGTCGAAGCTGCTCACGCTAGACTGTACCCCGATGCCCCACCCCTCCGTTTGTGTCGTGGAGCAAACGTTCGCTACCGTGTCCGTACTCGTTTCGGACCCGATACTCTGGTTCCAATGTCCCCTCGTGAATACCGTCGTGCTGTTCTGCCTGCCCTCGATGGACGTGCTGCCGCTTCTGGCGCTGGTGACGCTATGGCTCCTGGCGCTCCGGACTTCTGTGAGGATGAGGCTCACTCACATCGTGCCTGTGCCCGCTGGGGACTGGGCGCTCCATTGAGGCCTGTATACGTGGCACTGGGCCGTGATGCTGTTAGAGGCGGACCCGCTGAATTGAGAGGCCCTCGTCGTGAATTCTGTGCTAGGGCTCTGCTCGAACCCGATGGAGATGCTCCTCCTTTGGTACTCCGTGACGACGCCGATGCTGGTCCTCCCCCACAAATTCGCTGGGCTAGTGCTGCTGGACGTGCTGGTACTGTATTGGCTGCTGCTGGCGGTGGCGTTGAAGTTGTTGGTACTGCCGCTGGACTCGCTACACCTCCCCGCCGTGAACCTGTAGACATGGATGCTGAACTCGAGGATGATGACGACGGATTGTTCGGAGAG SEQ ID NO: 127 = construct RS1.6CACCACCACCACCACCATCATAGGCGTAGACGTGCTCCTAGACGTCGTTCTGCCGCTAGTGACTCTTCCAAATCTGGCTCTTCTTCATCTGCCTCTTCCGCTTCATCTTCGGCCTCATCGTCCTCTTCGGCATCCGCTTCGAGTAGTGATGATGATGATGACGACGACGCTGCTAGAGCCCCCGCTTCTGCTGCCGACCACGCTGCTGGCGGAACTTTGGGAGCCGACGACGAGGAGGCGGGAGTTCCTGCTCGTGCCCCGGGAGCTGCTCCGAGGCCTTCTCCACCCCGTGCTGAACCTGCTCCGGCTAGAACACCGGCCGCTACTGCTGGTAGACTGGAGCGTAGACGTGCCCGTGCTGCTGTGGCTGGTAGAGATGCTACTGGCCGCTTCACTGCTGGCCGTCCTAGACGTGTTGAACTGGACGCCGATGCTGCTTCTGGTGCTTTCTACGCCCGTTACCGTGATGGTTACGTGTCTGGTGAACCTTGGCCTGGCGCTGGTCCACCTCCGCCCGGACGTGTACTCTACGGTGGATTGGGCGATTCTCGCCCTGGTCTGTGGGGCGCTCCGGAGGCTGAGGAGGCTAGAGCCCGTTTCGAGGCTTCTGGTGCCCCTGCTCCTGTTTGGGCTCCTGAATTGGGCGACGCTGCTCAACAATACGCCCTCATCACACGCTTGCTGTACACTCCCGACGCCGAGGCTATGGGATGGCTCCAAAACCCTAGAGTTGCCCCTGGTGATGTTGCTCTGGATCAGGCTTGTTTCCGTATCTCCGGCGCTGCTCGTAACTCTTCTTCGTTCATCTCCGGTTCTGTGGCTAGAGCTGTGCCTCACTTGGGATACGCCATGGCCGCTGGACGTTTCGGCTGGGGACTGGCTCATGTTGCTGCCGCTGTAGCAATGTCTAGACGCTACGACCGTGCTCAAAAAGGATTCTTGCTCACGTCACTGAGGCGTGCTTACGCCCCTTTGTTGGCCCGTGAAAACGCTGCCCTCACTGGCGCCCGTACCCCCGATGACGGTGGCGACGCCAACCGCCACGATGGTGATGATGCTAGAGGCAAACCCGCTGCCGCTGCTGCTCCTTTGCCCTCTGCCGCCGCTTCCCCTGCCGATGAACGTGCTGTTCCTGCCGGTTACGGTGCCGCTGGTGTGTTGGCTGCTTTGGGACGCTTGAGTGCTGCCCCGGCTAGTGCCCCCGCTGGTGCCGATGACGATGACGATGACGATGGTGCTGGCGGAGGCGGTGGCGGTAGACGTGCTGAGGCTGGACGTGTTGCTGTTGAATGCCTGGCTGCCTGTAGAGGAATCTTGGAGGCTCTGGCCGAGGGATTCGACGGAGACTTGGCGGCTGTACCGGGACTGGCGGGAGCGAGGCCTGCCGCTCCACCTCGCCCCGGTCCTGCTGGTGCTGCCGCTCCTCCTCATGCCGACGCTCCTAGACTCCGTGCTTGGCTCCGTGAACTCCGTTTCGTTCGTGACGCTTTGGTTCTGATGAGACTGAGAGGCGACTTGAGAGTGGCTGGAGGATCCGAGGCTGCTGTTGCTGCTGTCCGTGCTGTTTCTTTGGTTGCTGGTGCTTTGGGCCCTGCTTTGCCGAGATCTCCCCGTTTGTTGTCGAGTGCCGCCGCTGCTGCCGCCGATTTGTTGTTCCAAAACCAATCCCTCCGCCCTCTGCTCGCCGACACTGTTGCCGCTGCCGATTCTCTGGCTGCTCCGGCTTCTGCCCCACGTGAAGCTCGTAAACGTAAATCACCCGCTCCGGCTCGTGCTCCCCCTGGTGGCGCCCCTAGACCCCCTAAAAAATCCCGTGCCGATGCCCCTAGACCTGCTGCTGCTCCCCCCGCTGGTGCTGCTCCCCCCGCTCCCCCTACTCCCCCCCCACGCCCACCTCGTCCCGCTGCCCTCACACGCCGTCCTGCTGAGGGACCCGATCCACAAGGCGGCTGGCGTAGACAACCTCCTGGCCCATCCCATACACCGGCACCATCTGCCGCTGCTTTGGAGGCTTACTGTGCTCCTCGTGCTGTGGCTGAACTCACCGATCATCCGCTGTTCCCTGCTCCCTGGCGTCCCGCCCTCATGTTCGATCCTAGAGCTTTGGCTTCCTTGGCCGCTCGTTGTGCTGCCCCTCCCCCTGGCGGTGCTCCGGCTGCTTTCGGTCCTCTCCGTGCCTCTGGTCCACTCCGCCGTGCCGCTGCCTGGATGAGACAAGTTCCCGACCCTGAGGATGTTAGAGTTGTGATCTTGTACTCGCCCTTGCCTGGCGAGGATTTGGCCGCTGGTAGAGCTGGCGGTGGCCCCCCTCCTGAATGGTCTGCTGAACGTGGTGGTTTGTCTTGCTTGTTGGCCGCCCTGGGAAACCGTCTGTGTGGTCCTGCTACTGCTGCTTGGGCTGGAAACTGGACTGGCGCTCCCGATGTTTCTGCTCTCGGTGCTCAAGGAGTTTTGCTGCTCTCTACTCGTGACTTGGCATTCGCTGGAGCTGTTGAATTCCTGGGACTCTTGGCTGGCGCTTGTGATAGGAGACTCATCGTCGTAAACGCTGTGAGAGCTGCCGATTGGCCTGCCGATGGTCCTGTTGTGTCTCGTCAACACGCTTACTTGGCTTGTGAAGTGTTGCCCGCTGTCCAATGTGCTGTTCGCTGGCCTGCTGCTCGTGATCTGAGGCGTACTGTTCTGGCTAGTGGTCGTGTTTTCGGACCTGGTGTTTTCGCTCGTGTCGAAGCTGCTCACGCTAGACTGTACCCCGATGCCCCACCCCTCCGTTTGTGTCGTGGAGCAAACGTTCGCTACCGTGTCCGTACTCGTTTCGGACCCGATACTCTGGTTCCAATGTCCCCTCGTGAATACCGTCGTGCTGTTCTGCCTGCCCTCGATGGACGTGCTGCCGCTTCTGGCGCTGGTGACGCTATGGCTCCTGGCGCTCCGGACTTCTGTGAGGATGAGGCTCACTCACATCGTGCCTGTGCCCGCTGGGGACTGGGCGCTCCATTGAGGCCTGTATACGTGGCACTGGGCCGTGATGCTGTTAGAGGCGGACCCGCTGAATTGAGAGGCCCTCGTCGTGAATTCTGTGCTAGGGCTCTGCTCGAACCCGATGGAGATGCTCCTCCTTTGGTACTCCGTGACGACGCCGATGCTGGTCCTCCCCCACAAATTCGCTGGGCTAGTGCTGCTGGACGTGCTGGTACTGTATTGGCTGCTGCTGGCGGTGGCGTTGAAGTTGTTGGTACTGCCGCTGGACTCGCTACACCTCCCCGCCGTGAACCTGTAGACATGGATGCTGAACTCGAGGATGATGACGACGGATTGTTCGGAGAGTAA SEQ ID NO: 128 = construct RS1.7ATGAGTGCCGAACAGCGTAAAAAGAAAAAAACCACCACCACGACCCAAGGACGTGGAGCTGAAGTTGCTATGGCGGATGAGGATGGAGGCCGCTTGAGAGCTGCTGCTGAGACTACTGGAGGACCTGGATCACCGGACCCTGCCGATGGACCCCCCCCTACACCAAACCCCGATCGTAGACCGGCTGCTAGACCTGGATTCGGATGGCATGGAGGACCCGAGGAAAACGAGGACGAGGCGGACGACGCCGCTGCCGACGCCGACGCCGATGAGGCTGCCCCTGCTTCTGGAGAGGCGGTAGACGAACCTGCTGCCGATGGAGTTGTTAGCCCTAGGCAATTGGCTTTGTTGGCGAGCATGGTAGACGAGGCTGTGAGAACAATCCCTTCCCCTCCCCCTGAACGTGATGGAGCACAAGAGGAGGCGGCTAGGAGTCCCTCACCACCCCGTACACCTTCTATGAGAGCGGATTACGGCGAGGAAAACGACGACGACGACGATGATGATGACGACGATGATCGTGATGCCGGACGCTGGGTTAGGGGACCTGAAACCACTTCTGCTGTCCGTGGAGCATACCCCGATCCTATGGCGAGTTTGAGCCCTAGACCACCTGCCCCGAGGAGACACCACCACCACCACCATCATAGGCGTAGACGTGCTCCTAGACGTCGTTCTGCCGCTAGTGACTCTTCCAAATCTGGCTCTTCTTCATCTGCCTCTTCCGCTTCATCTTCGGCCTCATCGTCCTCTTCGGCATCCGCTTCGAGTAGTGATGATGATGATGACGACGACGCTGCTAGAGCCCCCGCTTCTGCTGCCGACCACGCTGCTGGCGGAACTTTGGGAGCCGACGACGAGGAGGCGGGAGTTCCTGCTCGTGCCCCGGGAGCTGCTCCGAGGCCTTCTCCACCCCGTGCTGAACCTGCTCCGGCTAGAACACCGGCCGCTACTGCTGGTAGACTGGAGCGTAGACGTGCCCGTGCTGCTGTGGCTGGTAGAGATGCTACTGGCCGCTTCACTGCTGGCCGTCCTAGACGTGTTGAACTGGACGCCGATGCTGCTTCTGGTGCTTTCTACGCCCGTTACCGTGATGGTTACGTGTCTGGTGAACCTTGGCCTGGCGCTGGTCCACCTCCGCCCGGACGTGTACTCTACGGTGGATTGGGCGCCCGTACCCCCGATGACGGTGGCGACGCCAACCGCCACGATGGTGATGATGCTAGAGGCAAACCCGCTGCCGCTGCTGCTCCTTTGCCCTCTGCCGCCGCTTCCCCTGCCGATGAACGTGCTGTTCCTGCCGGTTACGGTGCCGCTGGTGTGTTGGCTGCTTTGGGACGCTTGAGTGCTGCCCCGGCTAGTGCCCCCGCTGGTGCCGATGACGATGACGATGACGATGGTGCTGGCGGAGGCGGTGGCGGTAGACGTGCTGAGGCTGGACGTGTTGCTGTTGAATGCCTGGCTGCCTGTAGAGGAATCTTGGAGGCTCTGGCCGAGGGATTCGACGGAGACTTGGCGGCTGTACCGGGACTGGCGGGAGCGAGGCCTGCCGCTCCACCTCGCCCCGGTCCTGCTGGTGCTGCCGCTCCTCCTCATGCCGACGCTCCTAGACTCCGTGCTTGGCTCCGTGAACTCCGTTTCGTTCGTGACGCTTTGGTTCTGATGAGACTGAGAGGCGACTTGAGAGTGGCTGGAGGATCCGAGGCTGCTGTTGCTGCTGTCCGTGCTGTTTCTTTGGTTGCTGGTGCTTTGGGCCCTGCTTTGCCGAGATCTCCCCGTTTGTTGTCGAGTGCCGCCGCTGCTGCCGCCGATTTGTTGTTCCAAAACCAATCCCTCCGCCCTCTGCTCGCCGACACTGTTGCCGCTGCCGATTCTCTGGCTGCTCCGGCTTCTGCCCCACGTGAAGCTCGTAAACGTAAATCACCCGCTCCGGCTCGTGCTCCCCCTGGTGGCGCCCCTAGACCCCCTAAAAAATCCCGTGCCGATGCCCCTAGACCTGCTGCTGCTCCCCCCGCTGGTGCTGCTCCCCCCGCTCCCCCTACTCCCCCCCCACGCCCACCTCGTCCCGCTGCCCTCACACGCCGTCCTGCTGAGGGACCCGATCCACAAGGCGGCTGGCGTAGACAACCTCCTGGCCCATCCCATACACCGGCACCATCTGCCGCTGCTTTGGAGGCTTACTGTGCTCCTCGTGCTGTGGCTGAACTCACCGATCATCCGCTGTTCCCTGCTCCCTGGCGTCCCGCCCTCATGTTCGATCCTAGAGCTTTGGCTTCCTTGGCCGCTCGTTGTGCTGCCCCTCCCCCTGGCGGTGCTCCGGCTGCTTTCGGTCCTCTCCGTGCCTCTGGTCCACTCCGCCGTGCCGCTGCCTGGATGAGACAAGTTCCCGACCCTGAGGATGTTAGAGTTGTGATCTTGTACTCGCCCTTGCCTGGCGAGGATTTGGCCGCTGGTAGAGCTGGCGGTGGCCCCCCTCCTGAATGGTCTGCTGAACGTGGTGGTTTGTCTTGCTTGTTGGCCGCCCTGGGAAACCGTCTGTGTGGTCCTGCTACTGCTGCTTGGGCTGGAAACTGGACTGGCGCTCCCGATGTTTCTGCTCTCGGTGCTCAAGGAGTTTTGCTGCTCTCTACTCGTGACTTGGCATTCGCTGGAGCTGTTGAATTCCTGGGACTCTTGGCTGGCGCTTGTGATAGGAGACTCATCGTCGTAAACGCTGTGAGAGCTGCCGATTGGCCTGCCGATGGTCCTGTTGTGTCTCGTCAACACGCTTACTTGGCTTGTGAAGTGTTGCCCGCTGTCCAATGTGCTGTTCGCTGGCCTGCTGCTCGTGATCTGAGGCGTACTGTTCTGGCTAGTGGTCGTGTTTTCGGACCTGGTGTTTTCGCTCGTGTCGAAGCTGCTCACGCTAGACTGTACCCCGATGCCCCACCCCTCCGTTTGTGTCGTGGAGCAAACGTTCGCTACCGTGTCCGTACTCGTTTCGGACCCGATACTCTGGTTCCAATGTCCCCTCGTGAATACCGTCGTGCTGTTCTGCCTGCCCTCGATGGACGTGCTGCCGCTTCTGGCGCTGGTGACGCTATGGCTCCTGGCGCTCCGGACTTCTGTGAGGATGAGGCTCACTCACATCGTGCCTGTGCCCGCTGGGGACTGGGCGCTCCATTGAGGCCTGTATACGTGGCACTGGGCCGTGATGCTGTTAGAGGCGGACCCGCTGAATTGAGAGGCCCTCGTCGTGAATTCTGTGCTAGGGCTCTGCTCGAACCCGATGGAGATGCTCCTCCTTTGGTACTCCGTGACGACGCCGATGCTGGTCCTCCCCCACAAATTCGCTGGGCTAGTGCTGCTGGACGTGCTGGTACTGTATTGGCTGCTGCTGGCGGTGGCGTTGAAGTTGTTGGTACTGCCGCTGGACTCGCTACACCTCCCCGCCGTGAACCTGTAGACATGGATGCTGAACTCGAGGATGATGACGACGGATTGTTCGGAGAG SEQ ID NO: 129 = construct RS1.8ATGAGTGCCGAACAGCGTAAAAAGAAAAAAACCACCACCACGACCCAAGGACGTGGAGCTGAAGTTGCTATGGCGGATGAGGATGGAGGCCGCTTGAGAGCTGCTGCTGAGACTACTGGAGGACCTGGATCACCGGACCCTGCCGATGGACCCCCCCCTACACCAAACCCCGATCGTAGACCGGCTGCTAGACCTGGATTCGGATGGCATGGAGGACCCGAGGAAAACGAGGACGAGGCGGACGACGCCGCTGCCGACGCCGACGCCGATGAGGCTGCCCCTGCTTCTGGAGAGGCGGTAGACGAACCTGCTGCCGATGGAGTTGTTAGCCCTAGGCAATTGGCTTTGTTGGCGAGCATGGTAGACGAGGCTGTGAGAACAATCCCTTCCCCTCCCCCTGAACGTGATGGAGCACAAGAGGAGGCGGCTAGGAGTCCCTCACCACCCCGTACACCTTCTATGAGAGCGGATTACGGCGAGGAAAACGACGACGACGACGATGATGATGACGACGATGATCGTGATGCCGGACGCTGGGTTAGGGGACCTGAAACCACTTCTGCTGTCCGTGGAGCATACCCCGATCCTATGGCGAGTTTGAGCCCTAGACCACCTGCCCCGAGGAGACACCACCACCACCACCATCATAGGCGTAGACGTGCTCCTAGACGTCGTTCTGCCGCTAGTGACTCTTCCAAATCTGGCTCTTCTTCATCTGCCTCTTCCGCTTCATCTTCGGCCTCATCGTCCTCTTCGGCATCCGCTTCGAGTAGTGATGATGATGATGACGACGACGCTGCTAGAGCCCCCGCTTCTGCTGCCGACCACGCTGCTGGCGGAACTTTGGGAGCCGACGACGAGGAGGCGGGAGTTCCTGCTCGTGCCCCGGGAGCTGCTCCGAGGCCTTCTCCACCCCGTGCTGAACCTGCTCCGGCTAGAACACCGGCCGCTACTGCTGGTAGACTGGAGCGTAGACGTGCCCGTGCTGCTGTGGCTGGTAGAGATGCTACTGGCCGCTTCACTGCTGGCCGTCCTAGACGTGTTGAACTGGACGCCGATGCTGCTTCTGGTGCTTTCTACGCCCGTTACCGTGATGGTTACGTGTCTGGTGAACCTTGGCCTGGCGCTGGTCCACCTCCGCCCGGACGTGTACTCTACGGTGGATTGGGCGATTCTCGCCCTGGTCTGTGGGGCGCTCCGGAGGCTGAGGAGGCTAGAGCCCGTTTCGAGGCTTCTGGTGCCCCTGCTCCTGTTTGGGCTCCTGAATTGGGCGACGCTGCTCAACAATACGCCCTCATCACACGCTTGCTGTACACTCCCGACGCCGAGGCTATGGGATGGCTCCAAAACCCTAGAGTTGCCCCTGGTGATGTTGCTCTGGATCAGGCTTGTTTCCGTATCTCCGGCGCTGCTCGTAACTCTTCTTCGTTCATCTCCGGTTCTGTGGCTAGAGCTGTGCCTCACTTGGGATACGCCATGGCCGCTGGACGTTTCGGCTGGGGACTGGCTCATGTTGCTGCCGCTGTAGCAATGTCTAGACGCTACGACCGTGCTCAAAAAGGATTCTTGCTCACGTCACTGAGGCGTGCTTACGCCCCTTTGTTGGCCCGTGAAAACGCTGCCCTCACTGGCGCCCGTACCCCCGATGACGGTGGCGACGCCAACCGCCACGATGGTGATGATGCTAGAGGCAAACCCGCTGCCGCTGCTGCTCCTTTGCCCTCTGCCGCCGCTTCCCCTGCCGATGAACGTGCTGTTCCTGCCGGTTACGGTGCCGCTGGTGTGTTGGCTGCTTTGGGACGCTTGAGTGCTGCCCCGGCTAGTGCCCCCGCTGGTGCCGATGACGATGACGATGACGATGGTGCTGGCGGAGGCGGTGGCGGTAGACGTGCTGAGGCTGGACGTGTTGCTGTTGAATGCCTGGCTGCCTGTAGAGGAATCTTGGAGGCTCTGGCCGAGGGATTCGACGGAGACTTGGCGGCTGTACCGGGACTGGCGGGAGCGAGGCCTGCCGCTCCACCTCGCCCCGGTCCTGCTGGTGCTGCCGCTCCTCCTCATGCCGACGCTCCTAGACTCCGTGCTTGGCTCCGTGAACTCCGTTTCGTTCGTGACGCTTTGGTTCTGATGAGACTGAGAGGCGACTTGAGAGTGGCTGGAGGATCCGAGGCTGCTGTTGCTGCTGTCCGTGCTGTTTCTTTGGTTGCTGGTGCTTTGGGCCCTGCTTTGCCGAGATCTCCCCGTTTGTTGTCGAGTGCCGCCGCTGCTGCCGCCGATTTGTTGTTCCAAAACCAATCCCTCCGCCCTCTGCTCGCCGACACTGTTGCCGCTGCCGATTCTCTGGCTGCTCCGGCTTCTACACCGGCACCATCTGCCGCTGCTTTGGAGGCTTACTGTGCTCCTCGTGCTGTGGCTGAACTCACCGATCATCCGCTGTTCCCTGCTCCCTGGCGTCCCGCCCTCATGTTCGATCCTAGAGCTTTGGCTTCCTTGGCCGCTCGTTGTGCTGCCCCTCCCCCTGGCGGTGCTCCGGCTGCTTTCGGTCCTCTCCGTGCCTCTGGTCCACTCCGCCGTGCCGCTGCCTGGATGAGACAAGTTCCCGACCCTGAGGATGTTAGAGTTGTGATCTTGTACTCGCCCTTGCCTGGCGAGGATTTGGCCGCTGGTAGAGCTGGCGGTGGCCCCCCTCCTGAATGGTCTGCTGAACGTGGTGGTTTGTCTTGCTTGTTGGCCGCCCTGGGAAACCGTCTGTGTGGTCCTGCTACTGCTGCTTGGGCTGGAAACTGGACTGGCGCTCCCGATGTTTCTGCTCTCGGTGCTCAAGGAGTTTTGCTGCTCTCTACTCGTGACTTGGCATTCGCTGGAGCTGTTGAATTCCTGGGACTCTTGGCTGGCGCTTGTGATAGGAGACTCATCGTCGTAAACGCTGTGAGAGCTGCCGATTGGCCTGCCGATGGTCCTGTTGTGTCTCGTCAACACGCTTACTTGGCTTGTGAAGTGTTGCCCGCTGTCCAATGTGCTGTTCGCTGGCCTGCTGCTCGTGATCTGAGGCGTACTGTTCTGGCTAGTGGTCGTGTTTTCGGACCTGGTGTTTTCGCTCGTGTCGAAGCTGCTCACGCTAGACTGTACCCCGATGCCCCACCCCTCCGTTTGTGTCGTGGAGCAAACGTTCGCTACCGTGTCCGTACTCGTTTCGGACCCGATACTCTGGTTCCAATGTCCCCTCGTGAATACCGTCGTGCTGTTCTGCCTGCCCTCGATGGACGTGCTGCCGCTTCTGGCGCTGGTGACGCTATGGCTCCTGGCGCTCCGGACTTCTGTGAGGATGAGGCTCACTCACATCGTGCCTGTGCCCGCTGGGGACTGGGCGCTCCATTGAGGCCTGTATACGTGGCACTGGGCCGTGATGCTGTTAGAGGCGGACCCGCTGAATTGAGAGGCCCTCGTCGTGAATTCTGTGCTAGGGCTCTGCTCGAACCCGATGGAGATGCTCCTCCTTTGGTACTCCGTGACGACGCCGATGCTGGTCCTCCCCCACAAATTCGCTGGGCTAGTGCTGCTGGACGTGCTGGTACTGTATTGGCTGCTGCTGGCGGTGGCGTTGAAGTTGTTGGTACTGCCGCTGGACTCGCTACACCTCCCCGCCGTGAACCTGTAGACATGGATGCTGAACTCGAGGATGATGACGACGGATTGTTCGGAGAG SEQ ID NO: 130 = His tag HHHHHH SEQ ID NO: 131 = Tag MSYYHHHHHHSEQ ID NO: 132 = Secretion Signal MKFLVNVALVFMVVYISYIYA SEQ ID NO: 133 =UL49.5ATGTCGTACTACCATCACCATCACCATCACATGACGGGGAAACCCGCAAGACTGGGCCGCTGGGTGGTGCTGTTGTTCGTCGCGCTCGTCGCGGGCGTGCCCGGGGAGCCGCCGAACGCGGCAGGCGCACGCGGCGTTATCGGGGACGCGCAATGCCGGGGCGACAGCGCCGGTGTGGTGTCCGTCCCGGGGGTCCTGGTGCCCTTTTATCTAGGCATGACCTCGATGGGCGTATGTATGATCGCGCACGTGTATCAGATATGCCAGCGGGCACTGGCCGCCGGGTCAGCCTGASEQ ID NO: 134 = UL10ATGGGACGCCGGGCCCCCAGGGGATCCCCCGAGGCCGCGCCGGGCGCCGACGTCGCGCCCGGGGCGCGGGCGGCGTGGTGGGTCTGGTGTGTGCAGGTGGCGACGTTCATCGTCTCGGCCATCTGCGTCGTGGGGCTCCTGGTGCTGGCCTCTGTGTTCCGGGACAGGTTTCCCTGCCTTTACGCCCCCGCGACCTCTTATGCGAAGGCGAACGCCACGGTCGAGGTGCGCGGGGGTGTAGCCGTCCCCCTCCGGTTGGACACGCAGAGCCTGCTGGCCACGTACGCAATTACGTCTACGCTGTTGCTGGCGGCGGCCGTGTACGCCGCGGTGGGCGCGGTGACCTCGCGCTACGAGCGCGCGCTGGATGCGGCCCGTCGCCTGGCGGCGGCCCGTATGGCGATGCCACACGCCACGCTAATCGCCGGAAACGTCTGCGCGTGGCTGTTGCAGATCACAGTCCTGCTGCTGGCCCACCGCATCAGCCAGCTGGCCCACCTTATCTACGTCCTGCACTTTGCGTGCCTCGTGTATCTCGCGGCCCATTTTTGCACCAGGGGGGTCCTGAGCGGGACGTACCTGCGTCAGGTTCACGGCCTGATTGACCCGGCGCCGACGCACCATCGTATCGTCGGTCCGGTGCGGGCAGTAATGACAAACGCCTTATTACTGGGCACCCTCCTGTGCACGGCCGCCGCCGCGGTCTCGTTGAACACGATCGCCGCCCTGAACTTCAACTTTTCCGCCCCGAGCATGCTCATCTGCCTGACGACGCTGTTCGCCCTGCTTGTCGTGTCGCTGTTGTTGGTGGTCGAGGGGGTGCTGTGTCACTACGTGCGCGTGTTGGTGGGCCCCCACCTCGGGGCCATCGCCGCCACCGGCATCGTCGGCCTGGCCTGCGAGCACTACCACACCGGTGGTTACTACGTGGTGGAGCAGCAGTGGCCGGGGGCCCAGACGGGAGTCCGCGTCGCCCTGGCGCTCGTCGCCGCCTTTGCCCTCGCCATGGCCGTGCTTCGGTGCACGCGCGCCTACCTGTATCACCGGCGACACCACACTAAATTTTTCGTGCGCATGCGCGACACCCGGCACCGCGCCCATTCGGCGCTTCGACGCGTACGCAGCTCCATGCGCGGTTCTAGGCGTGGCGGGCCGCCCGGAGACCCGGGCTACGCGGAAACCCCCTACGCGAGCGTGTCCCACCACGCCGAGATCGACCGGTATGGGGATTCCGACGGGGACCCGATCTACGACGAAGTGGCCCCCGACCACGAGGCCGAGCTCTACGCCCGAGTGCAACGCCCCGGGCCTGTGCCCGACGCCGAGCCCATTTACGACACCGTGGAGGGGTATGCGCCAAGGTCCGCGGGGGAGCCGGTGTACAGCACCGTTCGGCGATGGTAG SEQ ID NO: 135 =uracil DNA glycosylase encoded by UL2MKRARSRSPSPPSRPSSPFRTPPHGGSPRREVGAGILASDATSHVCIASHPGSGAGQPTRLAAGSAVQRRRPRGCPPGVMFSASTTPEQPLGLSGDATPPLPTSVPLDWAAFRRAFLIDDAWRPLLEPELANPLTARLLAEYDRRCQTEEVLPPREDVFSWTRYCTPDDVRVVIIGQDPYHHPGQAHGLAFSVRADVPVPPSLRNVLAAVKNCYPDARMSGRGCLEKWARDGVLLLNTTLTVKRGAAASHSKLGWDRFVGGVVQRLAARRPGLVFMLWGAHAQNAIRPDPRQHYVLKFSHPSPLSKVPFGTCQHFLAANRYLETRDIMPIDWSV SEQ ID NO: 136 =gL2 secreted v.2 encoded by construct UL1s v.2AGSQATEYVLRSVIAKEVGDILRVPCMRTPADDVSWRYEAPSVIDYARIDGIFLRYHCPGLDTFLWDRHAQRAYLVNPFLFAAGFLEDLSHSVFPADTQETTTRRALYKEIRDALGSRKQAVSHAPVRAGCVNFDYSRTRRCVGRRDLRPANTTSTWEPPVSSDDEASSQSKPLATQPPVLALSNAPPRRVSPTRGRRRHTRLRRN SEQ ID NO: 137 =UL1s v.2ATGAAGTTCCTCGTGAACGTGGCCCTGGTGTTCATGGTGGTGTACATCAGCTACATCTACGCCGCCGGGTCACAGGCAACCGAATATGTTCTTCGTAGTGTTATTGCCAAAGAGGTGGGGGACATACTAAGAGTGCCTTGCATGCGGACCCCCGCGGACGATGTTTCTTGGCGCTACGAGGCCCCGTCCGTTATTGACTATGCCCGCATAGACGGAATATTTCTTCGCTATCACTGCCCGGGGTTGGACACGTTTTTGTGGGATAGGCACGCCCAGAGGGCGTATCTTGTTAACCCCTTTCTCTTTGCGGCGGGATTTTTGGAGGACTTGAGTCACTCTGTGTTTCCGGCCGACACCCAGGAAACAACGACGCGCCGGGCCCTTTATAAAGAGATACGCGATGCGTTGGGCAGTCGAAAACAGGCCGTCAGCCACGCACCCGTCAGGGCCGGGTGTGTAAACTTTGACTACTCACGCACTCGCCGCTGCGTCGGGCGACGCGATTTACGGCCTGCCAACACCACGTCAACGTGGGAACCGCCTGTGTCGTCGGACGATGAAGCGAGCTCGCAGTCGAAGCCCCTCGCCACCCAGCCGCCCGTCCTCGCCCTTTCGAACGCCCCCCCACGGCGGGTCTCCCCGACGCGAGGTCGGCGCCGGCATACTCGCCTCCGACGCAACCATCACCATCACCATCACTGA SEQ ID NO: 138 =ICP4 internal fragment encoded by construct RS1.9(deletion of #391-544 and #786-821)MSAEQRKKKKTTTTTQGRGAEVAMADEDGGRLRAAAETTGGPGSPDPADGPPPTPNPDRRPAARPGFGWHGGPEENEDEADDAAADADADEAAPASGEAVDEPAADGVVSPRQLALLASMVDEAVRTIPSPPPERDGAQEEAARSPSPPRTPSMRADYGEENDDDDDDDDDDDRDAGRWVRGPETTSAVRGAYPDPMASLSPRPPAPRRHHHHHHHRRRRAPRRRSAASDSSKSGSSSSASSASSSASSSSSASASSSDDDDDDDAARAPASAADHAAGGTLGADDEEAGVPARAPGAAPRPSPPRAEPAPARTPAATAGRLERRRARAAVAGRDATGRFTAGRPRRVELDADAASGAFYARYRDGYVSGEPWPGAGPPPPGRVLYGGLGRTPDDGGDANRHDGDDARGKPAAAAAPLPSAAASPADERAVPAGYGAAGVLAALGRLSAAPASAPAGADDDDDDDGAGGGGGGRRAEAGRVAVECLAACRGILEALAEGFDGDLAAVPGLAGARPAAPPRPGPAGAAAPPHADAPRLRAWLRELRFVRDALVLMRLRGDLRVAGGSEAAVAAVRAVSLVAGALGPALPRSPRLLSSAAAAAADLLFQNQSLRPLLADTVAAADSLAAPASAAAPPAGAAPPAPPTPPPRPPRPAALTRRPAEGPDPQGGWRRQPPGPSHTPAPSAAALEAYCAPRAVAELTDHPLFPAPWRPALMFDPRALASLAARCAAPPPGGAPAAFGPLRASGPLRRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGAGDAMAPGAPDFCEDEAHSHRACARWGLGAPLRPVYVALGRDAVRGGPAELRGPRREFCARALLEPDGDAPPLVLRDDADAGPPPQIRWASAAGRAGTVLAAAGGGVEVVGTAAGLATPPRREPVDMDAELEDDDDGLFGE SEQ ID NO: 139 =ICP4 internal fragment encoded by construct RS1.10(deletion of #391-508 and #786-821)MSAEQRKKKKTTTTTQGRGAEVAMADEDGGRLRAAAETTGGPGSPDPADGPPPTPNPDRRPAARPGFGWHGGPEENEDEADDAAADADADEAAPASGEAVDEPAADGVVSPRQLALLASMVDEAVRTIPSPPPERDGAQEEAARSPSPPRTPSMRADYGEENDDDDDDDDDDDRDAGRWVRGPETTSAVRGAYPDPMASLSPRPPAPRRHHHHHHHRRRRAPRRRSAASDSSKSGSSSSASSASSSASSSSSASASSSDDDDDDDAARAPASAADHAAGGTLGADDEEAGVPARAPGAAPRPSPPRAEPAPARTPAATAGRLERRRARAAVAGRDATGRFTAGRPRRVELDADAASGAFYARYRDGYVSGEPWPGAGPPPPGRVLYGGLGAMSRRYDRAQKGFLLTSLRRAYAPLLARENAALTGARTPDDGGDANRHDGDDARGKPAAAAAPLPSAAASPADERAVPAGYGAAGVLAALGRLSAAPASAPAGADDDDDDDGAGGGGGGRRAEAGRVAVECLAACRGILEALAEGFDGDLAAVPGLAGARPAAPPRPGPAGAAAPPHADAPRLRAWLRELRFVRDALVLMRLRGDLRVAGGSEAAVAAVRAVSLVAGALGPALPRSPRLLSSAAAAAADLLFQNQSLRPLLADTVAAADSLAAPASAAAPPAGAAPPAPPTPPPRPPRPAALTRRPAEGPDPQGGWRRQPPGPSHTPAPSAAALEAYCAPRAVAELTDHPLFPAPWRPALMFDPRALASLAARCAAPPPGGAPAAFGPLRASGPLRRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGAGDAMAPGAPDFCEDEAHSHRACARWGLGAPLRPVYVALGRDAVRGGPAELRGPRREFCARALLEPDGDAPPLVLRDDADAGPPPQIRWASAAGRAGTVLAAAGGGVEVVGTAAGLATPPRREPVDMDAELEDDDDGLFGE SEQ ID NO: 140 = construct RS1.9ATGAGTGCCGAACAGCGTAAAAAGAAAAAAACCACCACCACGACCCAAGGACGTGGAGCTGAAGTTGCTATGGCGGATGAGGATGGAGGCCGCTTGAGAGCTGCTGCTGAGACTACTGGAGGACCTGGATCACCGGACCCTGCCGATGGACCCCCCCCTACACCAAACCCCGATCGTAGACCGGCTGCTAGACCTGGATTCGGATGGCATGGAGGACCCGAGGAAAACGAGGACGAGGCGGACGACGCCGCTGCCGACGCCGACGCCGATGAGGCTGCCCCTGCTTCTGGAGAGGCGGTAGACGAACCTGCTGCCGATGGAGTTGTTAGCCCTAGGCAATTGGCTTTGTTGGCGAGCATGGTAGACGAGGCTGTGAGAACAATCCCTTCCCCTCCCCCTGAACGTGATGGAGCACAAGAGGAGGCGGCTAGGAGTCCCTCACCACCCCGTACACCTTCTATGAGAGCGGATTACGGCGAGGAAAACGACGACGACGACGATGATGATGACGACGATGATCGTGATGCCGGACGCTGGGTTAGGGGACCTGAAACCACTTCTGCTGTCCGTGGAGCATACCCCGATCCTATGGCGAGTTTGAGCCCTAGACCACCTGCCCCGAGGAGACACCACCACCACCACCATCATAGGCGTAGACGTGCTCCTAGACGTCGTTCTGCCGCTAGTGACTCTTCCAAATCTGGCTCTTCTTCATCTGCCTCTTCCGCTTCATCTTCGGCCTCATCGTCCTCTTCGGCATCCGCTTCGAGTAGTGATGATGATGATGACGACGACGCTGCTAGAGCCCCCGCTTCTGCTGCCGACCACGCTGCTGGCGGAACTTTGGGAGCCGACGACGAGGAGGCGGGAGTTCCTGCTCGTGCCCCGGGAGCTGCTCCGAGGCCTTCTCCACCCCGTGCTGAACCTGCTCCGGCTAGAACACCGGCCGCTACTGCTGGTAGACTGGAGCGTAGACGTGCCCGTGCTGCTGTGGCTGGTAGAGATGCTACTGGCCGCTTCACTGCTGGCCGTCCTAGACGTGTTGAACTGGACGCCGATGCTGCTTCTGGTGCTTTCTACGCCCGTTACCGTGATGGTTACGTGTCTGGTGAACCTTGGCCTGGCGCTGGTCCACCTCCGCCCGGACGTGTACTCTACGGTGGATTGGGCCGTACCCCCGATGACGGTGGCGACGCCAACCGCCACGATGGTGATGATGCTAGAGGCAAACCCGCTGCCGCTGCTGCTCCTTTGCCCTCTGCCGCCGCTTCCCCTGCCGATGAACGTGCTGTTCCTGCCGGTTACGGTGCCGCTGGTGTGTTGGCTGCTTTGGGACGCTTGAGTGCTGCCCCGGCTAGTGCCCCCGCTGGTGCCGATGACGATGACGATGACGATGGTGCTGGCGGAGGCGGTGGCGGTAGACGTGCTGAGGCTGGACGTGTTGCTGTTGAATGCCTGGCTGCCTGTAGAGGAATCTTGGAGGCTCTGGCCGAGGGATTCGACGGAGACTTGGCGGCTGTACCGGGACTGGCGGGAGCGAGGCCTGCCGCTCCACCTCGCCCCGGTCCTGCTGGTGCTGCCGCTCCTCCTCATGCCGACGCTCCTAGACTCCGTGCTTGGCTCCGTGAACTCCGTTTCGTTCGTGACGCTTTGGTTCTGATGAGACTGAGAGGCGACTTGAGAGTGGCTGGAGGATCCGAGGCTGCTGTTGCTGCTGTCCGTGCTGTTTCTTTGGTTGCTGGTGCTTTGGGCCCTGCTTTGCCGAGATCTCCCCGTTTGTTGTCGAGTGCCGCCGCTGCTGCCGCCGATTTGTTGTTCCAAAACCAATCCCTCCGCCCTCTGCTCGCCGACACTGTTGCCGCTGCCGATTCTCTGGCTGCTCCGGCTTCTGCTGCTGCTCCCCCCGCTGGTGCTGCTCCCCCCGCTCCCCCTACTCCCCCCCCACGCCCACCTCGTCCCGCTGCCCTCACACGCCGTCCTGCTGAGGGACCCGATCCACAAGGCGGCTGGCGTAGACAACCTCCTGGCCCATCCCATACACCGGCACCATCTGCCGCTGCTTTGGAGGCTTACTGTGCTCCTCGTGCTGTGGCTGAACTCACCGATCATCCGCTGTTCCCTGCTCCCTGGCGTCCCGCCCTCATGTTCGATCCTAGAGCTTTGGCTTCCTTGGCCGCTCGTTGTGCTGCCCCTCCCCCTGGCGGTGCTCCGGCTGCTTTCGGTCCTCTCCGTGCCTCTGGTCCACTCCGCCGTGCCGCTGCCTGGATGAGACAAGTTCCCGACCCTGAGGATGTTAGAGTTGTGATCTTGTACTCGCCCTTGCCTGGCGAGGATTTGGCCGCTGGTAGAGCTGGCGGTGGCCCCCCTCCTGAATGGTCTGCTGAACGTGGTGGTTTGTCTTGCTTGTTGGCCGCCCTGGGAAACCGTCTGTGTGGTCCTGCTACTGCTGCTTGGGCTGGAAACTGGACTGGCGCTCCCGATGTTTCTGCTCTCGGTGCTCAAGGAGTTTTGCTGCTCTCTACTCGTGACTTGGCATTCGCTGGAGCTGTTGAATTCCTGGGACTCTTGGCTGGCGCTTGTGATAGGAGACTCATCGTCGTAAACGCTGTGAGAGCTGCCGATTGGCCTGCCGATGGTCCTGTTGTGTCTCGTCAACACGCTTACTTGGCTTGTGAAGTGTTGCCCGCTGTCCAATGTGCTGTTCGCTGGCCTGCTGCTCGTGATCTGAGGCGTACTGTTCTGGCTAGTGGTCGTGTTTTCGGACCTGGTGTTTTCGCTCGTGTCGAAGCTGCTCACGCTAGACTGTACCCCGATGCCCCACCCCTCCGTTTGTGTCGTGGAGCAAACGTTCGCTACCGTGTCCGTACTCGTTTCGGACCCGATACTCTGGTTCCAATGTCCCCTCGTGAATACCGTCGTGCTGTTCTGCCTGCCCTCGATGGACGTGCTGCCGCTTCTGGCGCTGGTGACGCTATGGCTCCTGGCGCTCCGGACTTCTGTGAGGATGAGGCTCACTCACATCGTGCCTGTGCCCGCTGGGGACTGGGCGCTCCATTGAGGCCTGTATACGTGGCACTGGGCCGTGATGCTGTTAGAGGCGGACCCGCTGAATTGAGAGGCCCTCGTCGTGAATTCTGTGCTAGGGCTCTGCTCGAACCCGATGGAGATGCTCCTCCTTTGGTACTCCGTGACGACGCCGATGCTGGTCCTCCCCCACAAATTCGCTGGGCTAGTGCTGCTGGACGTGCTGGTACTGTATTGGCTGCTGCTGGCGGTGGCGTTGAAGTTGTTGGTACTGCCGCTGGACTCGCTACACCTCCCCGCCGTGAACCTGTAGACATGGATGCTGAACTCGAGGATGATGACGACGGATTGTTCGGAGAGSEQ ID NO: 141 = construct RS1.10ATGAGTGCCGAACAGCGTAAAAAGAAAAAAACCACCACCACGACCCAAGGACGTGGAGCTGAAGTTGCTATGGCGGATGAGGATGGAGGCCGCTTGAGAGCTGCTGCTGAGACTACTGGAGGACCTGGATCACCGGACCCTGCCGATGGACCCCCCCCTACACCAAACCCCGATCGTAGACCGGCTGCTAGACCTGGATTCGGATGGCATGGAGGACCCGAGGAAAACGAGGACGAGGCGGACGACGCCGCTGCCGACGCCGACGCCGATGAGGCTGCCCCTGCTTCTGGAGAGGCGGTAGACGAACCTGCTGCCGATGGAGTTGTTAGCCCTAGGCAATTGGCTTTGTTGGCGAGCATGGTAGACGAGGCTGTGAGAACAATCCCTTCCCCTCCCCCTGAACGTGATGGAGCACAAGAGGAGGCGGCTAGGAGTCCCTCACCACCCCGTACACCTTCTATGAGAGCGGATTACGGCGAGGAAAACGACGACGACGACGATGATGATGACGACGATGATCGTGATGCCGGACGCTGGGTTAGGGGACCTGAAACCACTTCTGCTGTCCGTGGAGCATACCCCGATCCTATGGCGAGTTTGAGCCCTAGACCACCTGCCCCGAGGAGACACCACCACCACCACCATCATAGGCGTAGACGTGCTCCTAGACGTCGTTCTGCCGCTAGTGACTCTTCCAAATCTGGCTCTTCTTCATCTGCCTCTTCCGCTTCATCTTCGGCCTCATCGTCCTCTTCGGCATCCGCTTCGAGTAGTGATGATGATGATGACGACGACGCTGCTAGAGCCCCCGCTTCTGCTGCCGACCACGCTGCTGGCGGAACTTTGGGAGCCGACGACGAGGAGGCGGGAGTTCCTGCTCGTGCCCCGGGAGCTGCTCCGAGGCCTTCTCCACCCCGTGCTGAACCTGCTCCGGCTAGAACACCGGCCGCTACTGCTGGTAGACTGGAGCGTAGACGTGCCCGTGCTGCTGTGGCTGGTAGAGATGCTACTGGCCGCTTCACTGCTGGCCGTCCTAGACGTGTTGAACTGGACGCCGATGCTGCTTCTGGTGCTTTCTACGCCCGTTACCGTGATGGTTACGTGTCTGGTGAACCTTGGCCTGGCGCTGGTCCACCTCCGCCCGGACGTGTACTCTACGGTGGATTGGGCGCAATGTCTAGACGCTACGACCGTGCTCAAAAAGGATTCTTGCTCACGTCACTGAGGCGTGCTTACGCCCCTTTGTTGGCCCGTGAAAACGCTGCCCTCACTGGCGCCCGTACCCCCGATGACGGTGGCGACGCCAACCGCCACGATGGTGATGATGCTAGAGGCAAACCCGCTGCCGCTGCTGCTCCTTTGCCCTCTGCCGCCGCTTCCCCTGCCGATGAACGTGCTGTTCCTGCCGGTTACGGTGCCGCTGGTGTGTTGGCTGCTTTGGGACGCTTGAGTGCTGCCCCGGCTAGTGCCCCCGCTGGTGCCGATGACGATGACGATGACGATGGTGCTGGCGGAGGCGGTGGCGGTAGACGTGCTGAGGCTGGACGTGTTGCTGTTGAATGCCTGGCTGCCTGTAGAGGAATCTTGGAGGCTCTGGCCGAGGGATTCGACGGAGACTTGGCGGCTGTACCGGGACTGGCGGGAGCGAGGCCTGCCGCTCCACCTCGCCCCGGTCCTGCTGGTGCTGCCGCTCCTCCTCATGCCGACGCTCCTAGACTCCGTGCTTGGCTCCGTGAACTCCGTTTCGTTCGTGACGCTTTGGTTCTGATGAGACTGAGAGGCGACTTGAGAGTGGCTGGAGGATCCGAGGCTGCTGTTGCTGCTGTCCGTGCTGTTTCTTTGGTTGCTGGTGCTTTGGGCCCTGCTTTGCCGAGATCTCCCCGTTTGTTGTCGAGTGCCGCCGCTGCTGCCGCCGATTTGTTGTTCCAAAACCAATCCCTCCGCCCTCTGCTCGCCGACACTGTTGCCGCTGCCGATTCTCTGGCTGCTCCGGCTTCTGCTGCTGCTCCCCCCGCTGGTGCTGCTCCCCCCGCTCCCCCTACTCCCCCCCCACGCCCACCTCGTCCCGCTGCCCTCACACGCCGTCCTGCTGAGGGACCCGATCCACAAGGCGGCTGGCGTAGACAACCTCCTGGCCCATCCCATACACCGGCACCATCTGCCGCTGCTTTGGAGGCTTACTGTGCTCCTCGTGCTGTGGCTGAACTCACCGATCATCCGCTGTTCCCTGCTCCCTGGCGTCCCGCCCTCATGTTCGATCCTAGAGCTTTGGCTTCCTTGGCCGCTCGTTGTGCTGCCCCTCCCCCTGGCGGTGCTCCGGCTGCTTTCGGTCCTCTCCGTGCCTCTGGTCCACTCCGCCGTGCCGCTGCCTGGATGAGACAAGTTCCCGACCCTGAGGATGTTAGAGTTGTGATCTTGTACTCGCCCTTGCCTGGCGAGGATTTGGCCGCTGGTAGAGCTGGCGGTGGCCCCCCTCCTGAATGGTCTGCTGAACGTGGTGGTTTGTCTTGCTTGTTGGCCGCCCTGGGAAACCGTCTGTGTGGTCCTGCTACTGCTGCTTGGGCTGGAAACTGGACTGGCGCTCCCGATGTTTCTGCTCTCGGTGCTCAAGGAGTTTTGCTGCTCTCTACTCGTGACTTGGCATTCGCTGGAGCTGTTGAATTCCTGGGACTCTTGGCTGGCGCTTGTGATAGGAGACTCATCGTCGTAAACGCTGTGAGAGCTGCCGATTGGCCTGCCGATGGTCCTGTTGTGTCTCGTCAACACGCTTACTTGGCTTGTGAAGTGTTGCCCGCTGTCCAATGTGCTGTTCGCTGGCCTGCTGCTCGTGATCTGAGGCGTACTGTTCTGGCTAGTGGTCGTGTTTTCGGACCTGGTGTTTTCGCTCGTGTCGAAGCTGCTCACGCTAGACTGTACCCCGATGCCCCACCCCTCCGTTTGTGTCGTGGAGCAAACGTTCGCTACCGTGTCCGTACTCGTTTCGGACCCGATACTCTGGTTCCAATGTCCCCTCGTGAATACCGTCGTGCTGTTCTGCCTGCCCTCGATGGACGTGCTGCCGCTTCTGGCGCTGGTGACGCTATGGCTCCTGGCGCTCCGGACTTCTGTGAGGATGAGGCTCACTCACATCGTGCCTGTGCCCGCTGGGGACTGGGCGCTCCATTGAGGCCTGTATACGTGGCACTGGGCCGTGATGCTGTTAGAGGCGGACCCGCTGAATTGAGAGGCCCTCGTCGTGAATTCTGTGCTAGGGCTCTGCTCGAACCCGATGGAGATGCTCCTCCTTTGGTACTCCGTGACGACGCCGATGCTGGTCCTCCCCCACAAATTCGCTGGGCTAGTGCTGCTGGACGTGCTGGTACTGTATTGGCTGCTGCTGGCGGTGGCGTTGAAGTTGTTGGTACTGCCGCTGGACTCGCTACACCTCCCCGCCGTGAACCTGTAGACATGGATGCTGAACTCGAGGATGATGACGACGGATTGTTCGGAGAG

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Thus, for example, reference to “a cell” includes reference toone or more cells known to those skilled in the art, and so forth.Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. The invention includes embodiments in whichexactly one member of the group is present in, employed in, or otherwiserelevant to a given product or process. The invention includesembodiments in which more than one, or all of the group members arepresent in, employed in, or otherwise relevant to a given product orprocess. Furthermore, it is to be understood that the inventionencompasses all variations, combinations, and permutations in which oneor more limitations, elements, clauses, descriptive terms, etc., fromone or more of the listed claims is introduced into another claim. Forexample, any claim that is dependent on another claim can be modified toinclude one or more limitations found in any other claim that isdependent on the same base claim. Furthermore, where the claims recite acomposition, it is to be understood that methods of using thecomposition for any of the purposes disclosed herein are included, andmethods of making the composition according to any of the methods ofmaking disclosed herein or other methods known in the art are included,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the invention, or aspects ofthe invention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is noted that the term“comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or sub-rangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anyantigen, any method of administration, any prophylactic and/ortherapeutic application, etc.) can be excluded from any one or moreclaims, for any reason, whether or not related to the existence of priorart.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

Other Embodiments

Those of ordinary skill in the art will readily appreciate that theforegoing represents merely certain preferred embodiments of theinvention. Various changes and modifications to the procedures andcompositions described above can be made without departing from thespirit or scope of the present invention, as set forth in the followingclaims.

What is claimed is:
 1. An immunogenic composition comprising apharmaceutically-acceptable carrier, an adjuvant, and a nucleic acidcomprising a nucleotide sequence that encodes a polypeptide comprisingan amino acid sequence at least 90% identical to SEQ ID NO:1, whereinthe polypeptide has an internal deletion of amino acids 390-544 of SEQID NO:1 and has an internal deletion of amino acids 786-820 of SEQ IDNO:1.
 2. The immunogenic composition of claim 1, wherein the polypeptidelacks 1-20 amino acids at one or both termini of SEQ ID NO:1.
 3. Theimmunogenic composition of claim 1, wherein the immunogenic compositioncomprises at least one additional nucleic acid having a nucleotidesequence that encodes a polypeptide having an amino acid sequence atleast 85% identical to the amino acid sequence of SEQ ID NOS: 1, 3, 5,38, or
 138. 4. The immunogenic composition of claim 3, wherein said atleast one additional nucleic acid encodes a polypeptide having an aminoacid sequence at least 85% identical to SEQ ID NO:
 3. 5. The immunogeniccomposition of claim 1, wherein the adjuvant is a protein.
 6. Theimmunogenic composition of claim 5, wherein the protein is a cytokine.7. The immunogenic composition of claim 1, wherein the adjuvant is anucleic acid encoding a cytokine.
 8. The immunogenic composition ofclaim 6, wherein the cytokine is an interleukin.
 9. The immunogeniccomposition of claim 8, wherein the interleukin is interleukin-12. 10.An immunogenic composition comprising: a) a pharmaceutically-acceptablecarrier, b) a nucleotide sequence that encodes a polypeptide comprisingan amino acid sequence at least 90% identical to SEQ ID NO:1, whereinthe polypeptide has an internal deletion of amino acids 390-544 of SEQID NO:1 and has an internal deletion of amino acids 786-820 of SEQ IDNO:1; and c) at least one additional nucleic acid having a nucleotidesequence that encodes a polypeptide having SEQ ID NOS: 1, 3, 5, 38, or138, or an immunogenic fragment thereof.
 11. The immunogenic compositionof claim 10, wherein the at least one additional nucleic acids encodepolypeptides having SEQ ID NOS: 1 and 3, or an immunogenic fragmentthereof.
 12. The immunogenic composition of claim 1, wherein the nucleicacid is cDNA.
 13. The immunogenic composition of claim 1, wherein thenucleic acid is DNA.
 14. The immunogenic composition of claim 1, whereinthe nucleic acid is a portion of a plasmid.
 15. A treatment regimencomprising: (i) a first immunogenic composition comprising apharmaceutically-acceptable carrier and a nucleic acid comprising anucleotide sequence that encodes a polypeptide comprising at least oneof the polypeptides having an amino acid sequence at least 90% identicalto SEQ ID NO:1, wherein the polypeptide has an internal deletion ofamino acids 390-544 of SEQ ID NO:1 and has an internal deletion of aminoacids 786-820 of SEQ ID NO:1; and (ii) a boost formulation to beadministered subsequently comprising a polypeptide comprising an aminoacid sequence at least 90% identical to SEQ ID NO:1, wherein thepolypeptide has an internal deletion of amino acids 390-544 of SEQ IDNO:1 and has an internal deletion of amino acids 786-820 of SEQ ID NO:1,a nucleic acid comprising a nucleotide sequence that encodes apolypeptide comprising an amino acid sequence at least 90% identical toSEQ ID NO:1, wherein the polypeptide has an internal deletion of aminoacids 390-544 of SEQ ID NO:1 and has an internal deletion of amino acids786-820 of SEQ ID NO:1, or a combination thereof.
 16. The treatmentregimen of claim 15, wherein the boost formulation further comprises apolypeptide having an amino acid sequence at least 85% identical to theamino acid sequence of SEQ ID NO:1 or 3 or a nucleic acid having anucleotide sequence that encodes a polypeptide having an amino acidsequence at least 85% identical to the amino acid sequence of SEQ ID NO:1 or 3, or a combination thereof.
 17. The treatment regimen of claim 15,wherein the boost formulation further comprises a polypeptide having theamino acid sequence of SEQ ID NO:
 2. 18. The treatment regimen of claim15, wherein the boost formulation further comprises a polypeptide havingan amino acid sequence at least 85% identical to the amino acid sequenceof SEQ ID NO: 136 or a nucleic acid having a nucleotide sequence thatencodes a polypeptide having an amino acid sequence at least 85%identical to the amino acid sequence of SEQ ID NO: 3, or a combinationthereof.
 19. The treatment regimen of claim 15, wherein the boostformulation comprises a polypeptide having an amino acid sequence atleast 90% identical to SEQ ID NO:1, wherein the polypeptide has aninternal deletion of amino acids 390-544 of SEQ ID NO:1 and has aninternal deletion of amino acids 786-820 of SEQ ID NO:1.
 20. Thetreatment regimen of claim 15, wherein the boost formulation comprises anucleic acid comprising a nucleotide sequence that encodes a polypeptidehaving an amino acid sequence at least 90% identical to SEQ ID NO:1,wherein the polypeptide has an internal deletion of amino acids 390-544of SEQ ID NO:1 and has an internal deletion of amino acids 786-820 ofSEQ ID NO:1.
 21. The treatment regimen of claim 15, wherein the boostformulation comprises the same nucleic acids as the first immunogeniccomposition.
 22. A method for inhibiting genital herpes in a subject,comprising administering an effective amount of an immunogeniccomposition according to claim
 1. 23. The method of claim 22, whereinthe method inhibits genital herpes in a subject after a three doseregimen.
 24. The method of claim 22, wherein the subject is a human. 25.The method of claim 22, wherein the subject has not been previouslyinfected with HSV-2.
 26. A method for treating genital herpes in asubject, comprising administering an effective amount of an immunogeniccomposition according to claim
 1. 27. The method of claim 26, whereinthe method inhibits onset or reduces severity of genital herpessymptoms.
 28. The method of claim 27, wherein the method reduces thenumber of herpetic lesions.
 29. The method of claim 28, wherein themethod reduces the number of days a subject experiences herpeticlesions.
 30. The method of claim 26 or 27, wherein the method increasesthe IgG titer to one or more HSV-2 antigens.
 31. The method of claim 26or 27, wherein the method increases the T cell response to one or moreHSV-2 antigens.
 32. The method of claim 26 or 27, wherein the methodreduces the number of herpetic lesions at the onset of HSV-2 infection.33. The method of claim 26 or 27, wherein the method inhibits onset orreduces severity of genital herpes symptoms in a subject after a threedose regimen.
 34. The method of claim 26, wherein the subject is ahuman.
 35. The method of claim 26, wherein the subject has not beenpreviously infected with HSV-2.